Advances in Pharmacology Volume 3

ADVANCES IN

Pharmacology VOLUME 3

ADVANCES IN PHARMACOLOGY ADVISORY BOARD

D. BOVET Istituto Superiore d i Sanith Rome, Italy B. B. BRODIE National Heart Institute Bethesda, Maryland

J. H. BURN Oxford University Oxford, England A. CARLSSON Department of Pharmacology University of Gothenburg Gothenburg, Sweden

K. K. CHEN Lill y Research Laboratories Indianapolis, Indiana

J. F. DANIELLI Department of Biochemical Pharmacolog y School of Pharmacy Stat0 Univerrsity of New York at Buffalo Buffalo, New York

R. DOMENJOZ Pharmakologisches Institut Universitat Bonn Bonn, Germany B. N. HALPERN Ddpartement de Mddecine Expdrimentale College de France Paris, France

A. D. WELCH Department of Pharmacology Yale University Medical School New Haven, Connecticut

ADVANCES m IN

Pharmacology EDITED BY

SILVI0 GA RATTlN I

PARKHURST A. SHORE

Istituto di Ricerche Farmacologiche "Mario Negri" Milano, Italy

Department of Pharmacology The University of Texas Southwestern Medical School Dallas, Texas

VOLUME 3

1964

ACADEMIC P R E S S

New York and London

COPYRIGHT@ 1964, BY ACADEMIC PRESSINC. ALL RIGIHTS RESERVED. NO PART OF T H I S BOOK MAY BE REPRODUCED I N ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

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United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House, London W.1

LIBRARY OF CONGRESS CATALO~ CARD NUMBER: 61-18298

PRINTED I N THE UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME 3 Numbers in parentheses refer to the page on which the author’s contribution begins.

G. BIALY(285), Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts

G. A. DENEAU (267), Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan GUY M. EVERETT (83), Section of Neuropharmacology, Abbott Laboratories, North Chicago, Illinois; Department of Pharmacology and Therapeutics, Stritch School of Medicine, Loyola University, Chicago, Illinois ALEXANDER H. FRIEDMAN (83), Department of Pharmacology and Toxicology, University of Wisconsin Medical School, Madison, Wisconsin TAGE. MANSOUR(129) , Department of Pharmacology, Stanford University School of Medicine, Palo Alto, California E. MARLEY(167), Institute of Psychiatry, Maudsley Hospital, Londm, England G. PINCUS(285), Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts M. H. SEEVERS(267), Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan MARTINM. WINBURY ( l ) , Department of Pharmacology, WarnerLambert Research Institute, Morris Plains, New Jersey

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CONTENTS

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CONTRI~UTORS

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Experimental Approaches to the Development of Antianginal Drugs

MARTINM . WINBURY I . Introduction and Definition of the Problem . . . . . I1. Physiology and Biochemistry of the Heart . . . . . I11. Progress in the Treatment of Coronary Insufficiency . . . IV . Action of Nitrites . . . . . . . . . . . V. Approaches to Laboratory Evaluation of Antianginal Agents . VI . Other Potential Approaches . . . . . . . . References . . . . . . . . . . . .

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Pharmacological Aspects of Parkinsonism

ALEXANDER H . FRIEDMAN AND GUYM . EVERETT I . Introduction . . . . . . . . . I1. Nature of the Disease . . . . . . . I11. Conventional Pharmacotherapy of Parkinsonism IV . Histamine and Antihistamines . . . . . V . Screening Methods for Anti-Parkinson Drugs . VI . Drug-Induced Parkinsonism . . . . . VII . Rational Pharmacotherapy . . . . . . VIII . Summary . . . . . . . . . . References . . . . . . . . .

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The Pharmacology and Biochemistry of Parasitic He1minths TAGE . MANSOUR I . Introduction . . . . . . . . . . . . I1. The Pharmacology of the Neuromuscular System . . . I11. The Effect of Anthelmintic Agents on Neuromuscular System IV . Carbohydrate Metabolism . . . . . . . . . V . Control Mechanisms of Carbohydrate Metabolism . . . VI . Differences among Analogous Enzymes . . . . . . VII . Effect of Anthelmintic Agents on Carbohydrate Metabolism . VIII . Conclusion . . . . . . . . . . . . References . . . . . . . . . . . . vii

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CONTENTS

The Adrenergic System and Sympathomimetic Amines

E. MARLEY I. Introduction . . . . . . . . . . . . . . 168 I1. The Adrenergic System . . . . . . . . . . . 168 I11. Adrenergic Receptors . . . . . . . . . . . . 174 IV. Formation of Catecholamines . . . . . . . . . . 179 V . Preganglionic Nerves and Sympathin Secretion: Adrenal Gland . . 183 VI . Postganglionic Sympathetic Nerves and Sympathin Secretion: The Spleen . . . . . . . . . 198 VII . Peripheral Action of Sympathomimetic Amines: Structure-Activity Studies . . . . . . . . . 200 VIII . Sympathomimetic Amines and the Central Nervous System . . . 216 I X . Blockade of Adrenergic Neuron and Receptor . . . . . . 242 X . Inactivation of Amines . . . . . . . . . . . 248 X I . Conclusion . . . . . . . . . . . . . . 252 References . . . . . . . . . . . . . . 252

Pharmacological Aspects of Drug Dependence G. A . DENEAU AND M . H . SEEVERS I. Introduction . . . . . . . . . . . I1. Tolerance . . . . . . . . . . . . I11. Physical Dependence . . . . . . . . . IV . PBychogenic Dependence . . . . . . . . V. Attempts to Find Nondependence Producing Analgesics . VI . Antitussives . . . . . . . . . . . References . . . . . . . . . . .

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267 267 269 272 274 281 281

Drugs Used. in Control of Reproduction G PINCUS AND G. BIALY

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I . Introduction . . . . . . . . . I1. Reproductive Physiology . . . . . . I11. Male Fertility Control . . . . . . . IV. Drugs Used in Control of Female Reproduction . V. Future Problems and Possibilities in the Use of Drugs for Control of Reproduction . . . . References . . . . . . . . .

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SUBJECT INDEX.

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Experimental Approaches to the Development of Antianginal Drugs MARTINM . WINBURY Department of Pharmacology. Warner-Lambert Research Institute. M d Plains. New Jersey I. Introduction and Definition of the Problem . . . . . . . A. Coronary Insufficiency . . . . . . . . . . . . B . Response of the Anginal Patient to Exercise . . . . . . . C . Response of the Anginal Patient to Nitroglycerin . . . . . D. Conclusion . . . . . . . . . . . . . . . I1. Physiology and Biochemistry of the Heart . . . . . . . . A . General . . . . . . . . . . . . . . . B . Hemodynamic Factors Regulating Coronary Blood Flow . . . . . . . . C: Factors Influencing Myocardial Oxygen Consumption D . Neural and Humoral Factors . . . . . . . . . . E. Myocardial Metabolism in Situ . . . . . . . . . . I11. Progrem in the Treatment of Coronary Insufficiency . . . . . . A Thyroid Inhibition . . . . . . . . . . . . . B. Monoamine Oxidase Inhibitors . . . . . . . . . . C . Adrenergic-Blocking Agents . . . . . . . . . . D . Coronary Dilators Other Than Nitrites . . . . . . . . E. Miscellaneous Agents . . . . . . . . . . . . IV . Action of Nitrites . . . . . . . . . . . . . . A. Coronary Hemodynamic Actions . . . . . . . . . B . General Hemodynamic Actions . . . . . . . . . . C . Cardiac Metabolism . . . . . . . . . . . . D. Effect on Catecholamines . . . . . . . . . . . E. Comparison of Response in Normal and Anginal Individual . . . F Effect on Collateral Circulation . . . . . . . . . . G . Conclusion . . . . . . . . . . . . . . . V. Approaches to Laboratory Evaluation of Antianginal Agents . . . . A. Coronary Dilator Action . . . . . . . . . . . B. Total Metabolic Approach . . . . . . . . . . . C . Measurement of Myocardial Oxygen Tension . . . . . . D. Experimental Coronary I d c i e n c y Induced by Coronary Occlusion or Drugs . . . . . . . . . . . . . . . E. Experimental Coronary Insufficiency Induced by Atherosclerosis . F. Prolongation of Contractile Activity during Anoxia . . . . . G. Antagonism of Effect of Catecholamines on the Heart . . . . H. Arteriographic Techniques . . . . . . . . . . . I . Use of Radioactive Tracers . . . . . . . . . . . VI . Other Potential Approaches . . . . . . . . . . . A. Hemodynamic 1

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B. Metabolic

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1. Introduction and Definition of the Problem

For many years there has been tacit acceptance of the premise that coronary vasodilatation is the method of choice for the treatment of angina pectoris. This assumption originates from the early work with nitroglycerin which established the drug’s effectiveness in the management of angina pectoris in man and its coronary dilator action in animals. The “coronary vasodilator” concept has been carried over from the nitrites to other types of compounds and many non-nitrite coronary dilators have been prepared (Charlier, 1961) and studied clinically. An initial enthusiasm was reported for many of these compounds but with time negative results accumulated and, a t present, we are still left with the realization that based on objective clinical evidence, the nitrites are the only coronary vasodilators which show a beneficial effect. It is true that some of the earlier compounds developed may have produced coronary dilatation as a result of increased oxygen requirement, which we shall see is undersirable ; one of the more recently developed non-nitrite compounds, dipyridamole, increases the oxygen supply to the heart without markedly altering myocardial oxygen requirement but appears to be of little or no value in the treatment of angina pectoris (DeGraff and Lyon, 1963). By all criteria, this compound has the attributes of an agent which, theoretically, one would expect would have a beneficial effect in the treatment of angina pectoris (discussed in Section 111). It is obvious that there must be a re-evaluation of the concepts and theories regarding the mechanisms involved in angina pectoris and the approaches to the development of drugs for treatment of the disease. The use of coronary vasodilators necessitates the ability of the coronary bed to be able to dilate. Recent work has raised a question as to whether or not it is possible for drugs to increase the rate of coronary blood flow in the anginal patient (Gorlin e t al., 1959a; Rowe et al., 1961). Thus, there is a question about the exact mechanism of the antianginal effect of nitroglycerin. I n this chapter an attempt will be made to survey angina pectoris and the rational treatment from a physiological, biochemical, and pharmacological viewpoint. Methods for the laboratory evaluation of drugs will be reviewed objectively and potential new approaches considered. Angina pectoris is the syndrome associated with acute coronary insufficiency. It is merely the clinical sign of a basic pathophysiological problem resulting from atherosclerosis of the coronary arteries. There appears to be a relationship between myocardial ischemia resulting from “functional”

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coronary insufficiency and angina pectoris as well as the objective electrocardiographic abnormalities. The mechanism of the anginal pain has not been elucidated and it is not known if the pain originates in receptors in the ischemic myocardium or in the vessel wall. Present therapy is aimed at altering the circulation or obtunding the pain; however, the most desirable approach is one that improves the basic coronary insufficiency. The primary aim of this discussion is consideration of approaches to alleviate the coronary insufficiency. Therefore, when the term “antianginal” is used in this chapter, i t will refer to drugs aimed a t this objective.

A. CORONARY INSUFFICIENCY This can be defined as an imbalance between the available oxygen supply and the demand of the myocardial cells. This is a functional definition depending on the supply/demand ratio. The supply is dependent upon an adequate coronary blood flow, adequate oxygen saturation of arterial blood, effective capillary distribution and transfer of oxygen and substrate, and proper tissue utilization. The demand is related to the work load and metabolic status of the heart. Obviously, any decrease in the supply or increase in the demand will upset the balance and result in coronary insufficiency. At rest there is little difference between the coronary blood flow rate in the normal subject and in the anginal patient (Brachfeld e t al., 1959; Gorlin et al., 1959a; Gorlin, 196213). Under conditions producing an increased oxygen requirement of the heart the coronary flow in the normal patient can increase severalfold indicating a large “coronary reserve” ; however, in the anginal patient the flow can increase but little because of a limited coronary reserve and signs of coronary insufficiency may appear (pain and electrographic alterations). Thus there is nearly maximal vasodilatation a t rest in the anginal patient. The reduction in coronary reserve in the anginal patient is probably associated with a loss of the ability of the coronary vessels to dilate because of the underlying atherosclerosis. On this basis, coronary insufficiency results from a n increase in the demand for oxygen rather than a reduction in the supply. Studies in intact dogs and isolated rabbit hearts tend to confirm the fact that atherosclerosis can produce a reduction in coronary reserve (Karp e t al., 1960; Cross and Oblath, 1962; Melville and Varma, 1962). A spasm of the coronary artery would result in a decrease in the blood supply and thus produce coronary insufficiency. It seems unlikely that this is the case, but some still subscribe to this view because the nitrites can produce relaxation of an experimentally induced spasm in the animal (Modell, 1962). A recent report cites the case of one patient who had an anginal attack during coronary arteriography ; poor filling was observed

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in both the right and left coronary arteries (Gensini e t d.,1962). Administration of isosorbide dinitrate permitted good filling of the right coronary which was interpreted to be in spasm during the attack. Attempts to induce spasm of one coronary artery in the dog by occlusion of another coronary artery have been unsuccessful and it was concluded that there is no evidence for a vasoconstrictor reflex caused by occlusion (Wang et al., 1957). When embolization of the left descending or circumflex artery was produced with lycopodium spores and the caliber of the coronary vessels evaluated by arteriographic procedures, one group concluded that there was a spasm of the embolized and nonembolized artery (Guzman et al., 1962), while another group concluded that a spasm did not occur (West et al., 1962b). An impartial reviewer favored the view that a spasm did not occur (Schmidt, 1962).

B. RESPONSE OF

ANGINALPATIENT TO EXERCISE The physiological differences between the coronary circulation of the normal and anginal individual are readily demonstrated during exercise. This increases the work and oxygen requirement of the heart as a result of an increased cardiac output, elevation of blood pressure, and tachycardia. Under the stress of mild exercise the coronary blood flow, as measured by the nitrous oxide method, increased in both normal and anginal patients (Gorlin, 1962a; Messer and Neill, 1962; Wagman et al., 1962). Exercise also increased the clearance of N a P from the heart muscle of the anginal patient, indicating an increased capillary blood flow (Hollander et al., 1963). Much of the increased coronary blood flow in the anginal subject can be related to the rise in the perfusion pressure. A more sensitive parameter of the adequacy of the coronary circulation than flow alone is the myocardial oxygen extraction, as this is the only means of obtaining oxygen when flow is a limiting factor. I n normal subjects, extraction of oxygen from the arterial blood and coronary venous saturation are unchanged by exercise, but in anginal patients extraction increases progressively during exercise and coronary venous saturation falls (Gorlin, 1962a; Messer and Neill, 1962; Messer et al., 1962; Wagman et al., 1962). The increased oxygen extraction in the anginal patient is indicative of a n inadequate coronary reserve even though flow does increase. Another important difference between the two groups is the change in mechanical efficiency of the heart with a significant increase observed in the normal patients but little change in the anginal patient (Gorlin, 1962a; Messer and Neill, 1962). There is reasonably good correlation between the abnormal coronary hemodynamics (increased oxygen extraction and decreased sinus saturation) and positive electrocardioTHE

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graphic findings (STsegment depression) after exercise (Gorlin, 1962a,b; Messer and Neill, 1962; Messer et al., 1962; Wagman et al., 1962) ; an abnormal extraction was present in 82% of the anginal patients studied (Gorlin, 1962a; Messer and Neill, 1962; Messer et al., 1962). Anaerobic metabolism has been investigated in normal and anginal patients both a t rest and during exercise. The majority of studies revealed no apparent anaerobic metabolism based on “excess lactate” production (Gorlin, 1962a; Krasnow et d., 1962b; Messer and Neill, 1962; Wagman et al., 1962) or the lactate-pyruvate redox potential (Gudbjarnason et a?., 1962; Stock et al., 1962). In one study, 5 of 34 subjects produced excess lactate during exercise; 3 of these subjects had angina but several other anginal patients showed no excess lactate (Krasnow et al., 1962b). I n general, it can be concluded that anaerobic metabolism contributes only a small share of the total energy supply of the heart even during coronary insufficiency induced by exercise. The catecholamines increase the oxygen requirement of the heart by an increase in cardiac work and cardiac metabolism and may have a role in the production of coronary insdciency (Raab, 1956, 1962). Blood levels of norepinephrine and epinephrine were reported to be increased by exercise in anginal but not in normal patients (Gazes et al., 1959). Other investigators found no change in anginal or normal patients on exercise (Wagman et al., 1962). During exercise, emotional excitement, and other forms of stress there is stimulation of the sympathetic innervation of the heart resulting in release of endogenous norepinephrine which can exert a local effect on the myocardial cells, Therefore plasma levels may not be a good indicator of the role of catecholamines. ANGINALPATIENT TO NITROGLYCERIN Comparison of the responses of the normal and the anginal patient to nitroglycerin, illustrated in Fig. 1, suggests that the anginal individual has a “fixed” coronary resistance (Rowe, 1962). While the coronary flow increased 66% in the normal group the flow declined 16% in the anginal group (Brachfeld et al., 1959; Gorlin et al., 1959a). Coronary vascular resistance declined 42% in the normal group but there was no change in the anginal group. Blood pressure and cardiac work declined in both groups; cardiac oxygen consumption increased in normals but decreased in anginals. The clearance of N a P ’ did not increase after nitroglycerin in subjects with angina, indicating that capillary flow was also unchanged (Hollander et al., 1963). It has been suggested that angina induced by stress is associated with left ventricular failure. For example, during exercise there may be a rise

C. RESPONSEOF

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in pulmonary arterial pressure and left atrial pressure and an increase in diastolic heart size. Nitroglycerin will prevent or reverse these changes (Darby and Gebel, 1962; Stock et al., 1962). By means of coronary arteriographic techniques i t has been demonstrated that nitroglycerin and erythrityl tetranitrate increase the caliber

FIQ.1. Comparison of response of normal and anginal patient to nitroglycerin. MBP: mean blood pressure; CBF: coronary blood flow; CR: coronary resistance; A-V 01: arteriovenous oxygen difference; LVOz cons: left ventricular oxygen consumption; CO: cardiac output; LV work: left ventricular work; LV eff: left ventricular efficiency; HR: heart rate. Original data froin Brachfeld et al. (1959) and Gorlin et al. (1959a).

of the coronary arteries in anginal subjects (Likoff et al., 1962). However, the authors emphasize that no inference can be made that the increased caliber of the larger arteries is accompanied by an improvement in blood flow. It is important to realize that the smaller resistance vessels cannot be visualized and that these are the vessels that regulate the blood flow rate.

D. CONCLUSION The data that have been discussed above lead to the conclusion that the coronary bed in the anginal patient is adequate a t rest but is incapable of much dilatation. Therefore, the coronary reserve is seriously impaired. Any stress which increases the work and oxygen requirement of the heart will produce coronary insufficiency. It would appear that the beneficial effect of nitroglycerin is not associated with coronary dilatation but rather with the reduction in the work of the heart. However, it is conceivable that nitroglycerin may cause redistribution of blood to ischemic areas without producing a change in the total coronary blood flow rate.

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II. Physiology and Biochemistry of the Heart

A. GENERAL The heart is essentially an aerobic organ with a high rate of oxygen consumption. Myocardial arteriovenous oxygen difference (A-V 0,) is higher than that of any other organ and the oxygen extraction (A-V O,/A 0, X 100) has been estimated to be between 70 and 7576, compared with 22% for resting skeletal muscle and 25% for brain (Gorlin, 1962a). Under these circumstances, the oxygen supply to the myocardium can be considered essentially flow-limited since an increased oxygen extraction can yield only an additional 4 to 5 ~ 0 1 % .I n other organs additional oxygen can be obtained by increased extraction. During exercise, skeletal muscle will extract almost all of the available oxygen, as does cardiac muscle, but an “oxygen debt” can be contracted which is not the usual circumstance for the heart. For example, exercising skeletal muscle derives 10 to 30% of its energy anaerobically, compared with 5% for the heart during physical exercise (Gorlin, 1962a). Therefore, the factors that are involved in the regulation of coronary blood flow are of great importance in maintaining an adequate oxygen supply to the heart.

B. HEMODYNAMIC FACTORS REGULATING CORONARY BLOOD FLOW The blood flow through the coronary circulation (CBF) is related to the effective perfusion pressure divided by the resistance: CBF = effective perfusion pressure/resistance. These same physical principles apply to any vascular bed, but analysis of the interrelationship between various factors is different for the heart because the wall of the left ventricle, which develops the pressure head for perfusion of the coronary arteries, also offers phasic resistance to the coronary flow (Gregg, 1950). 1. Pressure Effective perfusion pressure is the difference between the pressure in the central coronary artery and the right atrium. A rise in perfusion pressure is invariably accompanied by a rise in coronary blood flow (Gregg, 1950; Alella et al., 1955; Berne, 1958; Rowe, 1962) ; however, the mechanism of the rise in coronary blood flow involves both physical and metabolic factors, the interrelationship of which will be discussed below. Only when the coronary arteries are perfused independently of the aorta can the direct relationship between pressure and flow be determined independently of metabolic factors (Berne, 1958) for if aortic pressure in-

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creases, the metabolic requirement of the heart increases because of an 1955; , Neil1 et al., 1963a). increased work level (Alella et a?. 2. Resistance Some of the factors which contribute to resistance are vessel tone (lumen size), extravascular support due to contraction of the myocardium, and relative duration of systole and diastole. The intrinsic portion of resistance is determined by the vessel tone regulated by the smooth muscles of the vessel walls. This is the more important portion of resistance and is involved in the autoregulation of the coronary circulation (Rowe, 1962). The arterioles on the afferent side of the capillary bed regulate vascular tone and the rate of flow out of the large arteries into the capillaries (Winsor and Hyman, 1961). Vessel tone can be influenced by endogenous and exogenous neurohumoral agents, metabolic products, nervous pathways to the coronary vessels, and by myocardial oxygen requirement (Gregg, 1950; Rowe, 1962). The latter is the most important determinant of vessel tone in the intact organism (Gorlin, 1962a). The extrinsic portion of resistance is a result of the mechanical or passive effect on flow due to the compression of the coronary vessels during ventricular systole (Gregg, 1950). Accordingly, there is a phasic increase in resistance during systole which is minimal during diastole. Approximately 75% of the flow occurs during diastole and 25% during systole (Gregg, 1962a). The phasic flow pattern emphasizes the interrelationship between the intrinsic and extrinsic portions of resistance and should be considered in analysis of drug action. With the onset of isometric contraction there is an abrupt decrease in left coronary inflow. I n open-chest dogs there may actually be backflow (Gregg, 1950, 1962a,b) but in normal unanesthetized dogs with measurement by an electromagnetic flowmeter no backflow is observed (Gregg, 1962b). After the aortic valves open and aortic pressure starts to rise, flow increases abruptly but declines somewhat during late systole. As isometric relaxation begins, flow again increases sharply to a new peak and declines gradually during diastole. On the other hand, outflow from the coronary sinus rises and falls smoothly with most flow occurring during systole. The inflow pattern is the important one since this relates to the supply to the capillaries which are compressed during systole. The importance of extravascular compression has been demonstrated in another way (Gregg and Sabiston, 1956; McKeever et al., 1958). The left coronary artery was perfused a t constant pressure from a reservoir with inflow and outflow measured continuously. During prolonged diastolic relaxation induced by vagal stimulation or ventricular fibrillation,

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the inflow and outflow invariably increased (Gregg and Sabiston, 1956; McKeever et al., 1958). It becomes obvious that any increase in systolic force or intraventricular pressure will increase the extravascular component of resistance. 3. Heart Rate

Changes in heart rate influence cardiac metabolism and affect flow indirectly (see Section 11, C) . However, changes in rate can influence the relative durations of systole and diastole; since diastolic flow accounts for 75% of the total, any alteration in the ratio systolic duration/diastolic duration will influence total coronary flow (Gregg, 1962b; Rowe, 1962). From a hemodynamic standpoint the important parameter is systolic time/minute versus diastolic time/minute. An increase in diastolic time a t the expense of systolic time should permit an increased rate of coronary flow. Other factors, such as viscosity of blood, also contribute to resistance but these are of minor importance except under unusual circumstances (Rowe, 1962). It is important to remember that resistance is a computed value relating flow to pressure under a particular circumstance and may or may not indicate changes in vascular tone. Nevertheless, the tone is the most important single variable in resistance. C. FACTORS INFLUENCING MYOCARDIAL OXYGENCONSUMPTION It has been well established that the coronary flow is correlated directly with the oxygen consumption (or requirement) of the heart (Alella et al., 1955; Katz, 1956; Berglund et al., 1957; Braunwald et al., 1958; Hashimoto et al., 1960; Gregg, 1962b; Rowe, 1962). If the oxygen supply is inadequate for the requirement there will be a reduction in vessel tone resulting in an increased flow. Thus, one can conclude that the 0, supply/02 demand ratio is the primary regulator of coronary flow from the metabolic standpoint (Gorlin, 1962a). I n fact, Katz (1956) suggested that hemodynamic factors play a minor role in the homeostasis of the coronary circulation in the absence of alterations of oxygen consumption. However, we should not lose sight of the fact that the mechanical influences involved in coronary flow, discussed previously, modify cardiac metabolism and in that way can influence coronary flow. That the coronary flow will be changed by alterations in oxygen content of the arterial blood has been demonstrated in the intact animal (Berne et al., 1957; Gregg, 1962b; Berne, 1963) and in the isolated rabbit heart (Guz et al., 1960). At normal perfusion pressure, a reduction in the oxygen content of the arterial blood is accompanied by a rise in coronary flow and a decline in the oxygen content of the coronary sinus

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blood (Berne et al., 1957; Scott et al., 1962). However, a t high perfusion pressures (greater than aortic pressure) the coronary flow is greater than normal and does not change with a decline in arterial oxygen content until the sinus content drops to less than 5.5 vol% ; below this level coronary flow increases as arterial and sinus oxygen content decline (Berne et al., 1957). Oxygen consumption does not vary except when the arterial content is exceedingly low (5 ~ 0 1 % )(Berne et al., 1957; Gregg, 1962b). Berne et al. (1957) concluded that the arterial oxygen content has no direct influence on the coronary arterioles when the oxygen supply is adequate. Only when sinus content declines below 5.5 vol% does coronary flow increase. Since the coronary venous blood reflects the changes in the cells of the myocardium it can be assumed that the oxygen tension of the tissues are of prime importance in the metabolic regulation of coronary flow (Berne et al., 1957). Katz (1956) concluded that coronary flow adjusts by a regulatory mechanism to maintain the A-V O2and venous oxygen content constant over the normal range of cardiac activity. The question arises as to whether or not myocardial hypoxia leads to the formation or release of vasodilator materials which could be involved in the autoregulation of the coronary circulation (Berne et al., 1957; Berne, 1963). Reoxygenated coronary sinus blood from normal, hypoxic, or hyperperfused hearts contained no vasoactive, inotropic, or chronotropic substance (Jelliffe et al., 1957). If any such materials were released by the tissues they were destroyed by the blood or by oxygenation. It has been suggested that adenosine may be involved in coronary autoregulation (Berne, 1963). During hypoxia of the isolated cat heart or hypoxemia of the intact dog there is a decrease in coronary vascular resistance and a release of inosine and hypoxanthine from the myocardium. These are metabolic degradation products of adenosine, which is a potent coronary dilator, and the amounts released, if considered as adenosine, could easily account for the coronary dilatation observed. The key factor in the autoregulation hypothesis of Berne (1963) is tissue oxygen tension. A decline in oxygen tension would result in breakdown of myocardial adenine nucleotides to form adenosine, which diffuses out of the cells and reaches the arterioles via interstitial fluid to produce arteriolar dilatation. Further experimental evidence is required to substantiate this hypothesis. 1. Hemodynamic Factors Influencing Oxygen Consumption.

The oxygen consumption and metabolism of the heart are adjusted to the heat production and useful work performed. Only a small part of the total energy liberated appears as useful mechanical work as evidenced by the low efficiency. The work of the heart is determined by the pressure generated (ventricular pressure) and the blood flow (stroke volume or

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cardiac output). Some have found a good correlation between oxygen consumption and cardiac work (Katz et al., 1955), whereas others have found a poor correlation (Neill et al., 1963a). Much of this divergence of correlation depends upon the type of experiment performed and the fact that the oxygen requirement for pressure work and flow work differ markedly. Oxygen consumption increases less than mechanical work when cardiac output is the predominant factor, but more than mechanical work when elevation of aortic pressure is involved (Alella et al.,1955; Braunwald et al., 1958; Sarnoff et aZ., 1958s; Katz et al., 1962; Neill et al., 1963a). Thus, when work is altered by increasing the resistance to outflow (aortic pressure) a t constant cardiac output, oxygen consumption increases markedly (175% increase in work, increase of Q4 178%) whereas increasing cardiac output at constant aortic pressure causes little change in oxygen consumption (696% increase in work, increase of Qo 53%) (Alella e t aZ., 1955; Braunwald et aZ., 1958; Sarnoff e t al., 1958a). Using the “isolated supported” heart preparation, Sarnoff et al. (1958a) found that the oxygen consumption of the heart was best correlated with the tension-time index, TTI (mean systolic pressure X duration of systole) under conditions of variations in aortic pressure and cardiac output. Recent studies by Neill et al. (1963a) in intact dogs showed that the PTI (mean systolic pressure x ejection period )( heart rate) was highly correlated with the oxygen consumption of the heart (correlation coefficient 0.93). The PTI/Qo, ratio was constant a t high and low PTI values, a t short or long ejection periods, and even under conditions of anemia in which the A-V 0, was reduced. Levine and Wagman (1962) point out that PTI is only one of the two components of myocardial tension; the other is the mean radius of the cardiac chamber. If we assume that the ventricle is a sphere, the law of Laplace applies as follows:

T =P d where T = total wall tension in dynes; P = intracavitary pressure in dynes/cm3; and r = radius in centimeters, Thus tension varies directly with pressure and with the square of the radius. At constant radius T will vary directly with PTI (and TTI) ; however, if during systole, the internal surface area (4Xr2) decreases more rapidly than P rises, T will decrease (Levine and Wagman, 1962). The importance of tension in determining oxygen consumption is more easily demonstrated in isolated cardiac muscle. As the resting tension level increases the portion of the total oxygen consumption during activity due to resting tension increases more rapidly than the portion due to activity (Lee, 1960). Under isotonic conditions (no tension) oxygen consumption did not vary with changes in the load (Whalen, 1962).

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Heart rate has an effect on coronary flow and oxygen consumption independent of changes in cardiac work (Katz, 1956; Laurent et al., 1956; Sarnoff et al., 1958a). At constant aortic pressure and cardiac output an increase in heart rate will increase oxygen consumption and thus lower mechanical efficiency (Laurent et al., 1956; Sarnoff et a,?., 1958a). However the PTI/Qo, ratio is independent of changes in heart rate (Neil1 et al., 1963a). A “stress adaptation mechanism,” described by Laurent et al. (1956) , was typified by an abnormal increase in coronary flow, a marked decline and a marked reduction in cardiac oxygen consumption even in A-V 02, though work was maintained a t a high level. This occurred when the normal oxygen supply/demand ratio was exceeded, as with a n increased load or excessive heart rate ; hypoxemia also brought on this mechanism but in this case coronary sinus blood became more and more unsaturated (Katz et al., 1955; Katz, 1956; Katz and Feinberg, 1958). After the stress adaptation mechanism became established, there was still an increase in cardiac oxygen consumption and coronary flow with an increase in heart rate, but the oxygen consumption was less at each level of heart rate than before even though work was greater. The stress adaptation mechanism lasted for more than 1 hour and was reversible. I n effect it was akin to shifting gears in a car. It is unlikely that the shift in metabolism involved a simple oxygen debt (Sarnoff et al., 1958~).Other possibilities are (1) use of storage products, (2) more efficient utilization of substrates, (3) more efficient conversion of energy to mechanical work, or (4) diversion of energy from synthesis and repair (resting state) to mechanical work (Katz and Feinberg, 1958; Sarnoff et al., 1958~).Another investigator suggested that a sudden release of anaerobic energy resulted when the oxygen supply became inadequate (Ballinger and Vollenweider, 1962). To my mind i t is necessary t o demonstrate that the stress adaptation mechanism exists in the intact animal since the results may be peculiar to the experimental design. The total oxygen consumption of the beating heart is a composite of resting oxygen consumption and activity oxygen consumption. The importance of each of these parameters was investigated in open-chest dogs with the left coronary artery perfused a t a constant pressure from an external source. The work of the heart was reduced to zero by vagal or potassium arrest, or by ventricular fibrillation (Berglund et al., 1957; McKeever et al., 1958). All of these procedures produced an initial increase in coronary flow (Berglund et al., 1957; McKeever e t al., 1958) and oxygen consumption (McKeever et al., 1958). This was rapidly followed by a decline in oxygen consumption to considerably below control (doing external work) values. During vagal or potassium arrest the oxygen

DEVELOPMENT OF ANTIANGINAL DRUGS

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consumption was about 25% of the activity level and during fibrillation about 50%; hemorrhage reduced oxygen consumption to about 35% of control values (McKeever et al., 1958). The basal oxygen consumption during fibrillation or hemorrhage was higher than that during vagal or potassium arrest (Berglund et al., 1957; McKeever et al., 1958). Similar results were obtained in the closed-chest dog; however, no difference was noted between arrest and fibrillation (Beuren et d.,1958). If the heart was stressed by high arterial pressure or administration of catecholamines there was a disproportionate rise in resting and active oxygen consumption with resting consumption accounting for 50% of the total (McKeever et al., 1958; Gregg, 1962a). In the potassium-arrested heart, norepinephrine decreased coronary flow but increased oxygen consumption (Berne, 1958). Presumably the resting oxygen consumption was used for maintenance of cellular integrity (selective membrane permeability) and production of a pool of high-energy phosphate which could be used for the contractile process; however, i t is difficult to explain the increase in the resting oxygen consumption produced by catecholamines or previous high work levels. In the case of the catecholamines i t is conceivable that inefficient metabolic pathways were stimulated. Computations of mechanical efficiency of the heart are usually based on the caloric equivalent of the total oxggen cmumptkm which includes both the resting and activity consumption. However, to determine true mechanical efficiency, only the energy required for the mechanical activity should be considered. When the resting metabolism is deducted from the total metabolism the efficiency of cardiac muscular activity increases to 37% for the dog and 39% for man (Gregg and Sabiston, 1956; Bing et al., 1958; Bing and Michal, 1959). Assuming that the resting metabolism does not vary greatly, it is easy to understand why the usual computed efficiency of the heart (about 25%) increases with an increased work level. This is true in the anesthetized open-chest dog whether the increased work level is due to a rise in cardiac output or in blood pressure (Katz et al., 1955). I n normal humans, the mechanical efficiency increases 30% during exercise while oxygen consumption increases 40% (Gorlin, 1962a; Messer and Neill, 1962). Similarly, the ratio of PTI/ oxygen consumption of the left ventricle increases 20% during exercise (Levine and Wagman, 1962). 2. Biochemical Factors Influencing Oxygen CmumpCion

It has been demonstrated that the utilization of certain substances by the heart is related to the level in the arterial blood (see Section 11,E, 2). Accordingly it is of interest to ascertain if oxygen consumption is related to the oxygen content of the arterial blood. Berne and co-workers (Berne

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et al., 1957; Berne, 1958) altered the oxygen supply to the heart of openchest dogs either by altering the rate of coronary flow or by altering the saturation of the arterial blood and found no change in oxygen consumption except in one experiment when the arterial content dropped below 5 ~ 0 1 % Using . the isolated rabbit heart, Guz et al. (1960) varied the oxygen content of the perfusion medium by using hemoglobin solutions of various concentrations that were equilibrated with a mixture of 3% CO, and 97% 02.Oxygen consumption and isometric contractile force remained constant, providing the oxygen content of the perfusion fluid did not decline below 2 ~ 0 1 % .On the other hand, Kats e t al. (1955) found that oxygen consumption was higher a t a given level of cardiac work as the arterial oxygen content increased. These conclusions were based on pooled data from individual experiments which show marked variability and the relationship between oxygen consumption and arterial supply may be spurious or a result of some other variables. I n intact dogs, Scott e t al. (1962) compared the effects of breathing 100, 10, and 5% oxygen mixtures. The 10% oxygen mixture produced an increase in cardiac work and oxygen consumption compared with 100% O2; under 5% oxygen cardiac work was again increased but oxygen consumption was reduced below that with 100% oxygen. Studies in anesthetized dogs with the left ventricular volume held constant by inflating a balloon therein suggested that cardiac oxygen consumption and left ventricular performance (heart rate x left ventricular pressure) were determined by the rate of coronary flow (Katz et al., 1962). The data available do not permit the conclusion that the oxygen consumption is related to the arterial content of oxygen or to the rate of coronary flow if the supply is adequate. Only when the supply is inadequate would a decline in cardiac oxygen consumption be expected. Raab has stressed the role of catecholamines in angina because of their effect on myocardial metabolism and oxygen consumption (Raab, 1956, 1962; Raab et al., 1962). The increased oxygen consumption is over and above that required for the additional work. Thus, the catecholamines cause wasteful metabolism with a reduction in cardiac efficiency (Raab, 1956,1962; Bing et al., 1960; Raab et al., 1962). The increase in myocardial oxygen consumption produced by catecholamines has been demonstrated by a number of different procedures. Injection of epinephrine, norepinephrine, and isoproterenol into the coronary circulation of the dog produced an increase in oxygen consumption and, usually, in coronary flow in the intact beating heart or in fibrillating or arrested heart (Eckstein et al., 1951 ; Berne, 1958; Hashimoto et al., 1960; Juhhss-Nagy and Szentivinyi, 1961 ; Winbury et al., 1962a). Similar effects were noted in intact cats following intravenous injection of epi-

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nephrine and norepinephrine; these changes were noted even if there was little change in cardiac work or heart rate (Popovich et al., 1956). Stimulation of the cardiac sympathetic nerves in the dog also produced an increase in oxygen consumption and coronary flow which was not always accompanied by a concomitant rise in heart rate, blood pressure, or aortic pulse (Eckstein et al., 1951; Juhhsz-Nagy and Ssentivinyi, 1961; Szentivhnyi and Juhhsz-Nagy, 1961; Gregg, 1962b). Chronic sympathectomy reduced coronary blood flow, heart rate, and myocardial oxygen consumption and was accompanied by a rise in left ventricular efficiency (Scott and Balourdas, 1959). Infusion of isoproterenol into normal man produced a rise in coronary flow and oxygen consumption together with an increase in stroke and cardiac index, heart rate, and mean systolic pressure (Krasnow et al., 1962a). The increase in oxygen consumption was proportional to the increase in PTI. Thus, PTI/Qc, was unchanged by the catecholamine. Thyroid hormone has an effect on myocardial oxygen consumption either by a direct action on myocardial function (work and heart rate) or metabolism or by sensitization of the myocardium to endogenously released catecholamines (Raab, 1956, 1962). It has been reported by therapy in patients with thyrotoxicosis caused Rowe et al. (1956) that a decrease in oxygen consumption, coronary flow, cardiac output, and left ventricular work. Gorlin (1962b), using the data of Rowe et al. (1956) to compute TTI, found that Il3' reduced this parameter but that the TTI/Qo, ratio was unchanged. Gudbjarnason et al. (1962) suggested that there is an uncoupling of oxidative phosphorylation in thyrotoxicosis.

D. NEURALAND HUMORAL FACTORS 1. F u n c t i m l Innervation of the COTOTZUTY Vessels

The heart receives both sympathetic and parasympathetic fibers which are involved in the central regulation of rate, conduction, contractility, and metabolism. The role of the sympathetic and parasympathetic systems in the regulation of the coronary circulation has been the subject of considerable controversy because many of the studies failed to consider the changes in myocardial metabolism, which can influence the coronary blood flow. Recently Szentivhnyi and Juhhsz-Nagy (Szentivhnyi and JuhhszNagy, 1959, 1961; Juhhsz-Nagy and Szentivhnyi, 1961) proposed a hypothesis for the functional innervation of the heart and coronary vessels which integrates the direct coronary and direct myocardial actions. The accelerator nerve (sympathetic) contains preganglionic fibers in addition to the postganglionic fibers. The postganglionic (C) fibers run directly to

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the muscle elements of the pacemaker system and of the myocardium, and influence heart rate, contractility, and myocardial metabolism. These postganglionic fibers do not affect the coronary arteries directly but can have an indirect effect by altering myocardial metabolism. The preganglionic ( B ) fibers of the sympathetic nerves do not form synapses in the stellate ganglion but synapse peripherally with adrenergic or cholinergic ganglia in or near the walls of the heart. The related postganglionic fibers innervate the coronary arteries directly with the sympathetic cholinergics producing dilation and sympathetic adrenergics producing constriction. These fibers do not innervate the muscle elements. The vagus nerve does not have any direct role in coronary innervation. The parasympathetic fibers synapse in the myocardium and the related postganglionic fibers go only to the pacemaker and the conducting system, influencing only rate and conduction, There is little effect on contractility, metabolism or coronary. circulation. Presumably the acetylcholine released a t these nerve terminals does not enter the coronary system but acts on specific cardiac tissue. I n some circumstances the vagus nerve contains preganglionic sympathetic fibers which innervate only the muscle cells. This hypothesis is supported by the work of Hashimoto e t al. (1960) using the fibrillating dog heart. 2. Effect of Sympathetic Stimulation and Catecholamines

Injection of catecholamines into the coronary circulation or sympathetic stimulation has been reported to produce constriction or dilatation, or both effects. The effect of sympathetic stimulation on coronary blood flow and other hemodynamic factors was analyzed by individual consecutive beats by Winbury and Green (1952). When coronary perfusion pressure was held constant there was an initial decline in coronary flow (constriction) followed in a short time by a marked increase (dilatation), but when perfusion pressure was permitted to rise with aortic pressure only an increase in coronary flow was observed. Computed coronary vascular resistance showed an initial increase followed by a decline in both types of experiments; heart rate and cardiac work increased. These effects were not blocked by atropine or ergotoxine. Eckstein et d. (1951) demonstrated that sympathetic stimulation with constant perfusion pressure produced coronary dilatation accompanied by a decline in A-V O2 and a rise in oxygen consumption. Berne (1958) noted that the dilatation was preceded by constriction as did Winbury and Green (1952). Szentivbnyi and Juhbss-Nagy (1959, 1961; Juhbsa-Nagy and Szentivbnyi, 1961) used a selective stimulation of the B and C fibers of the sympathetic nerves to separate the direct coronary effects from the in-

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direct metabolic effects. Stimulation of a branch of the cardiac sympathetic nerve of the dog with low voltages frequently resulted in coronary constriction without a change in heart rate, contraction or metabolism (Juhitsz-Nagy and Szentivhnyi, 1961). This effect was blocked by hexamethonium, ergotamine, or dibenamine, but not by atropine, indicating that preganglionic fibers of sympathetic adrenergic nerves were stimulated (JuhBsz-Nagy and Szentivhnyi, 1961; Szentivhyi and Juhhsz-Nagy, 1961). In the cat, stimulation of certain rami of the accelerator nerve resulted in coronary dilatation and sometimes in bradycardia; the dilatation was abolished by hexamethonium or atropine and enhanced by eserine (Szentivhnyi and Juhhsz-Nagy, 1959). These are preganglionic fibers of sympathetic cholinergic nerves. A similar pathway can be excited in some dogs by stimulation of the accelerator nerve with low parameters (JuhAss-Nagy and Szentivfinyi, 1961; Szentivhnyi and Juh fisz-Nagy, 1961) ; only vasodilatation was noted. This vasodilatation was not accompanied by any change in myocardial metabolism and, as in the cat, was abolished by hexamethonium or atropine, and enhanced by eserine. These sympathetic cholinergic nerves are interpreted to represent the true dilators of the coronaries (Szentivhnyi and Juhhsz-Nagy, 1961). When high voltages were used on the accelerator nerve in the dog there was an increase in cardiac contractility and rate, coronary flow, and oxygen consumption (Juhbz-Nagy and Szentivhnyi, 1961; Szentivhnyi and Juhhsz-Nagy, 1961). These effects were not abolished by hexamethonium, atropine, ergotamine, or dibenamine. These are postganglionic adrenergic fibers which go directly to the cardiac muscle but not to the coronaries. The vasodilation observed was secondary to the increased metabolism (oxygen consumption) (Szentivhnyi and Juhhss-Nag, 1959, 1961; Juhhsz-Nagy and Szentivhnyi, 1961). Since stimulation of these postganglionic adrenergic fibers always affects metabolism, most investigators failed to recognize the role of sympathetic preganglionic fibers which synapse with adrenergic constrictors or cholinergic dilators and have claimed that the coronaries do not have any independent vasomotor control. Catecholamines such as epinephrine, norepinephrine, and isoproterenol invariably produce coronary dilatation in the beating in situ or isolated heart (Eckstein et al., 1951; Winbury and Green, 1952; Berne, 1958; Hashimoto et al., 1960; Szentivhnyi and Juhhsz-Nagy, 1961). This is accompanied by an increase in cardiac Contractility (contractile force, stroke volume, or cardiac output), cardiac work, and oxygen consumption (Eckstein et al., 1951; Winbury and Green, 1952; Popovich et al., 1956; Berne, 1958; Hashimoto et al., 1960; Szentivhnyi and Juhfisz-Nagy, 1961). These effects are not antagonized by a-adrenergic-blocking agents (di-

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MARTIN M. WINBURY

benamine, ergotoxine, ergotamine) (Winbury and Green, 1952 ; Hashimoto e t aZ., 1960; Juhhsz-Nagy and SzentivLnyi, 1961; SzentivLnyi and Juhhsz-Nagy, 1961) ; however, Denison et al. (1956) reported that high doses of azapetine injected into the coronary artery block completely the increase in diastolic flow produced by epinephrine, norepinephrine, and isoproterenol. Berne (1958) and Hashimoto et al. (1960) demonstrated that epinephrine and norepinephrine have a direct vasoconstrictor effect in addition to the metabolic dilator action. I n the fibrillating dog heart perfused a t constant pressure, Berne (1958) observed that epinephrine and norepinephrine produce an immediate decline in coronaiy flow followed within a short time by an increase; oxygen consumption was not changed during vasoconstriction but increased during vasodilatation. When the fibrillating heart was arrested with potassium, norepinephrine produced only vasoconstriction even though oxygen consumption increased. Phasic flow measurements in the intact dog with the coronaries perfused a t constant pressure demonstrated that norepinephrine causes a temporary decline in diastolic flow before the rise (Berne, 1958). Hashimoto et al. (1960) reported that epinephrine, norepinephrine, and isoproterenol increase coronary flow and oxygen consumption in the fibrillating dog heart. After the &locking agent, dibenzylinc, the dilator effect of epinephrine and norepinephrine was increased but the metabolic effect was unchanged. After the &blocking agent, dichloroisoproterenol, isoproterenol had no effect, and epinephrine and norepinephrine produced a short constriction with no effect on metabolism. These results are in agreement with the hypothesis of SzentivLnyi and JuhBsz-Nagy (1961) that the only direct adrenergic effect on the coronaries is constriction. Preliminary studies by Winbury et al. (1962a) suggested that norepinephrine causes a reduction in “effective capillary flow” a t the time of vasodilatation and increased oxygen consumption. In these studies blood was pumped into the left coronary artery a t a constant rate with perfusion pressure as a measure of total resistance. Effective capillary flow was determined by RbS6 uptake of the myocardium; details of the procedure are given in Section V, I. Phasic coronary flow curves show that epinephrine, norepinephrine, isoproterenol, and sympathetic stimulation increase diastolic flow and decrease systolic flow (Denison et al., 1956; Berne, 1958; Denison and Green, 1958). The increase in diastolic flow is greater than the decrease in systolic flow, causing an increase in mean flow. The heart contains large catecholamine stores primarily in the form of norepinephrine (Raab, 1956) as a result of synthesis and accumulation. Sympathetic nerve stimulation will elicit discharge of norepinephrine from the adrenergic nerves directly into the myocardial effector cells (Raab,

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1962). Stimulation of cardiac sympathetic nerves is followed by an increase in myocardial norepinephrine and sympathetic denervation by a decrease (Raab, 1962). 3. Effect of Parasympathetic Stimulation and Acetylcholine There appears to be general agreement that stimulation of the vagus innervation of the heart has no effect on the coronary circulation independent of changes in heart rate (Winbury and Green, 1952; Schreiner et al., 1957; Denison and Green, 1958). When heart rate is controlled, vagus stimulation does not affect coronary blood flow or oxygen consumption. On the other hand, injection of acetylcholine into the coronary artery is always followed by coronary dilatation with no change in oxygen consumption (Winbury and Green, 1952; Schreiner et al., 1957; Berne, 1958; Juhhsz-Nagy and Szentivhnyi, 1961 ; Gregg, 1962b). Phasic flow measurements demonstrated a rise in both systolic and diastolic flow (Berne, 1958). It can be concluded that acetylcholine produces true coronary dilatation. The fact that vagal stimulation has no direct effect on the coronaries but that acetylcholine does is understandable in light of the hypothesis of Szentivhnyi and Juhhsz-Nagy (1959, 1961) that the parasympathetic fibers to the heart innervate only the pacemaker. Confirmation of this is provided by the fact that ventricular function is not affected by vagal stimulation but is depressed by injected acetylcholine (Schreiner et al., 1957). Injected acetylcholine mimics the vasodilator action of the preganglionic sympathetic cholinergic fibers. Coronary dilatation produced by acetylcholine and by stimulation of sympathetic cholinergics can be blocked by atropine (Winbury and Green, 1952; Szentivirnyi and JuhBszNagy, 1961).

E. MYOCARDIAL METABOLISM in Situ There are numerous studies on cardiac muscle slices, homogenates, etc., which have described enzymic pathways, substrate utilization, aerobic and anaerobic metabolism. In many cases i t is exceedingly difficult to relate the results obtained with these preparations to the results obtained on in situ intact preparations. Since the objective is to relate myocardial metabolism to the function of the heart, only in situ studies will be discussed. Substrate utilization, aerobic metabolism, and anaerobic metabolism will be considered. 1. General Considerations

It was mentioned previously that the heart has a high rate of oxygen uptake; consequently, extraction of oxygen from blood is nearly com-

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MARTIN M. WINBURY

plete. Under normal circumstances an increased oxygen requirement necessitates an increased coronary flow since the heart can contract only a small oxygen debt. When the coronary circulation is compromised as in coronary insdciency, the oxygen supply cannot keep pace with the demand and alterations in metabolism might be expected. The problem of specific substrates utilized for energy production has been studied in recent years by many groups. It has been demonstrated that the dog heart and human heart can use glucose, lactate, pyruvate, fatty acids, ketone bodies, and amino acids (Gregg and Sabiston, 1956; Bing et al., 1958, 1960; Bing, 1961; Stock et al., 1962). The utilization of these substrates is in direct relationship to the arterial level of each substance; however, there are thresholds below which a substrate will not be extracted by the heart and there is ‘(sparing” of one substrate by another (Gregg and Sabiston, 1956; Bing, 1961; Olson, 1962a,b; Shipp et al., 1962). A complication in the interpretation of the results is the fact that the uptake of a single substrate such as fatty acids or carbohydrates can account for more than 100% of t,he oxygen consumption indicating that there must be storage (Goodale and Hackel, 195313; Ballard e t al., 1960; Bing et al., 1960; Olson, 1962a,b; Shipp et al., 1962). This only serves to emphasize that uptake of a particular substrate by the heart does not indicate that the substrate is metabolized. 2. Substrate Uptake and Utilization Substrate uptake has been studied in dogs and man by determination of arteriovenous difference and coronary flow rate (A-V substrate difference, mg/ml blood X coronary flow, ml/minute = mg uptake/minute) . The share each substrate contributes to the total oxygen consumption can be computed on the basis of oxygen required for oxidation of substrate. Substrate utilization varies considerably under different dynamic circumstances (Ballard et al., 1960; Olson, 1962b). The extent to which each substrate contributes to cardiac energy production is dependent upon the arterial concentration, the state of nutrition, and the endocrine balance (Gregg and Sabiston, 1956; Bing et al., 1958; Olson, 1962b). During fasting or the postabsorptive state the heart shifts from predominantly carbohydrate utilization t o primarily fat utilization. Under postprandial conditions carbohydrates are the major substrates (Goodale and Hackel, 1953b; Gregg and Sabiston, 1956; Bing e t al., 1958; Ballard et al., 1960; Bing, 1961; Goto, 1962; Olson, 1962b). The respiratory quotient (RQ) of the fasting human or dog heart is about 0.80 and about 3/6 of the oxygen uptake can be accounted for by fatty acids and about 1/3 by carbohydrates (Bing and Michal, 1959; Bing, 1961; Olson, 1962b). On the other hand, after feeding or glucose infusion, the RQ of the heart

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is close to 1.0 and carbohydrates account for most of the oxygen utilization (Goodale and Hackel, 1953b; Olson, 1962b). According to Bing and co-workers (1958), the relative contributions of substrates to myocardial oxygen usage in the postabsorptive state is as follows: carbohydrates, 34.947% (glucose 17.9, pyruvate 0.54, and lactate 16.5) ; noncarbohydrates, 76.9% (fatty acids 67.0, amino acids 5.6, and ketones 4.3). The sum of these values accounts for more than 100%of the oxygen consumption, for the values are based on the assumption that the substrates taken up by the heart are completely oxidized. It is obvious that this is not the case and some is stored in the myocardium (Bing et al., 1958; Bing and Michal, 1959; Ballard et al., 1960). This is demonstrated by studies in which blood levels of fatty acids or glucose were raised and the contribution of each to the oxygen uptake exceeded 100% (Goodale and Hackel, 1953b; Bing et al., 1958). Olson (1962b) concluded that carbohydrates are the preferred substrates and are the determinants of the fuel used by the heart; the uptake of nonesterified fatty acids (NEFA) is regulated by the carbohydrate uptake. Glucose administration decreases the arterial level of fatty acids and cardiac extraction of NEFA (Bing et al., 1958). The interaction between palmitate, pyruvate, and acetoacetate was studied in the isolated perfused rat heart using Cl*-labeled palmitate (Olson, 1962a). When palmitate was the only substrate in the Krebs-Henseleit solution a high percentage was extracted, of which about 75% was oxidized to COz and accounted for 37% of the cardiac oxygen consumption. I n the presence of pyruvate or acetoacetate the uptake of palmitate was decreased 50% and oxidation to CO, was reduced to 25% of the value when palmitate was given alone. The uptake of pyruvate or acetoacetate was the same as if they were perfused alone. The carbohydrate contributed 7545% to the fuel of respiration and reduced the contribution of the fatty acid to 8-9%. The presence of pyruvate or acetoacetate shifted fatty acid from oxidation into a five- to sixfold increase in storage as neutral lipids in the heart. In contrast, other workers (Shipp et al., 1962), also using the perfused rat heart with labeled substrates, observed that when glucose and palmitate were present the fatty acid was oxidized in preference to glucose; the latter was converted to glycogen. There are definite threshold concentrations below which carbohydrates will not be extracted from arterial blood. In the dog these values are as follows: lactate, 2.56 mg%; pyruvate, 0.61 mg%; and glucose, 54.2 mg% (Goodale and Hackel, 1953b). The threshold for glucose in man has been estimated to be about 80 mg% (Bing et al., 1958; Bing, 1961). Above these values the extraction increases linearly with arterial concentration for lactate and pyruvate ; whereas with glucose, extraction increases linearly

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MARTIN M. WINBURY

up to about 100 mg% and then tends to become asymptotic (Goodale and Hackel, 195313; Olson, 1962b). At normal arterial concentrations the utilization of lactate is about that of glucose and is many times that of pyruvate (Goodale and Hackel, 1953b; Bing, 1961). For example, in the dog 66% of the oxygen extraction could be attributed to carbohydrates with the breakdown as follows: lactate, 34.1% ; pyruvate, 4.1% ; and glucose 27.1% (Goodale and Hackel, 1953b). However, if the arterial level of any one of these substrates is increased the proportion it contributes to the total carbohydrate uptake increases proportionately. Myocardial metabolism of fatty acids has been studied extensively in the dog and human. I n the fasting human the oxygen extraction ratio [ (0, equivalent of extracted fatty acidJmyocardial0, extraction) X 1001 for total fatty acids (TFA) averaged 145% (Ballard et al., 1960). The nonesterified fatty acids (NEFA) accounted for 42% of the total fatty acid extraction and the esterified portion made up the remaining 58% (Ballard et al., 1960; Bing, 1961). I n the fasting dog the TFA oxygen extraction ratio was 123% and NEFA accounted for 23% of the total extraction (Ballard et al., 1960; Bing, 1961). The iodine number of TFA was consistently higher in the coronary sinus blood than arterial blood, suggesting greater usage of more saturated fatty acids (Ballard et al., 1960). I n a recent study on open-chest dogs it was found that the arteriovenous differences for cholesterol, phospholipid, and TFA were quite variable and not statistically significant, but the arteriovenous difference for NEFA was significant (Goto, 1962). On the other hand, the isolated perfused rabbit heart can extract and oxidize NEFA bound to albumin as well as triglyceride fatty acids in lipoproteins (Gousios et al., 1962). The importance of the various long-chain fatty acids in the NEFA fraction has been analyzed and i t was found that the extraction of oleic acid was high compared with the other fatty acids (Bing, 1961; Carlsten et al., 1962; Goto, 1962). I n the human, extraction (in terms of moles/liter of blood) is 50 for oleic, 10 for stearic, and 2 for linoleic acids, respectively, indicating that oleic contributes considerably more to NEFA oxygen extraction ratio than stearic or lineolic (Carlsten et al., 1962). Other workers found the uptake of palmitic acid in man was also low. I n the dog oleic acid was the only long-chain fatty acid with a significant extraction; the other acids studied showed a nonsignificant positive extraction, or a negative extraction (Goto, 1962). These include lauric, myristic, palmitic, palmitoleic, stearic, linoleic, linolenic, and arachidic acids. 3. Anaerobic Metabolism There is considerable evidence that glycolysis can contribute to myocardial energy production under anaerobic conditions but there is a ques-

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tion as to whether glycolysis occurs under aerobic conditions. One group of investigators suggested that the normal dog heart has a slight “oxygen debt” on the basis of the fact that after arrest cardiac oxygen consumption did not decline for 10 to 15 seconds (Gregg and Sabiston, 1956; McKeever et al., 1958). On the other hand, another group found that when the work of the heart was reduced by lowering the outflow resistance there was a rapid readjustment of the oxygen consumption which was interpreted to indicate that an oxygen debt was ( a ) not present, ( b ) was present but that i t was repaid a t some other time, or (c) the debt remained constant despite the change in activity (Sarnoff et al., 1 9 5 8 ~ )If. an oxygen debt were present in the normal dog heart one might expect a rise in oxygen consumption with an increase in availability; however, no change in oxygen consumption was observed when arterial saturation varied between 5 and 19 vol% (Berne et al., 1957). Biochemical studies uniformly show that under aerobic conditions the heart of the dog and man utilizes lactate and certainly does not produce lactate under normal conditions (Bing and Michal, 1959; Huckabee, 1961; Gudbjarnason e t al., 1962; Stock et al., 1962; Neill et al., 1963a). Huckabee (1961) found that exercise produced by stimulating the hindlimb of the normal anesthetized dog resulted in “excess lactate” production by the heart and that anaerobic metabolism accounted for 12% of the energy production. This could not be confirmed in man since there was no increase in lactate production in normal or anginal individuals (Krasnow e t al., 1962b; Messer and Neill, 1962; Wagman et al., 1962). These investigators concluded t.hat anaerobic metabolism does not occur in the human heart even under the stress of exercise. A recent study by Neill and co-workers (1963b) in dogs showed that under control conditions the total energy output (external mechanical work and heat production) of the left ventricle (44.4 cal/min/100 gm) was approximately equal to the energy from oxidative metabolism (38.5 cal/min/100 gm).Mechanical work accounted for only about ‘/4 of the total energy output with the remainder being heat. Following injection of cyanide the total energy output (50.9 cal) was 4 times the energy available from substrate oxidation (11.4 cal) ; thus after cyanide anaerobic energy (39.5 cal) accounted for about 4/5 of the total. These changes were accompanied by excess lactate production. Mechanical energy now accounted for about ‘/z of the total energy output. Although these effects were of short duration they demonstrate that anaerobic energy production can be an important factor in maintaining cardiac activity under anaerobic conditions. I n studies on the effect of hypoxia on coronary flow the heart continued to function when the arterial oxygen saturation dropped to 5 vol% and the oxygen uptake of the heart was markedly

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reduced (Berne et al., 1957), indicating that anaerobic mechanisms were utilized. Many investigators have demonstrated that there is a net production of lactate (venous concentration higher than arterial) by the heart under hypoxic conditions and on this basis have concluded that anaerobic metabolism has occurred (Bing and Michal, 1959; Bing, 1961). This conclusion may not always be valid, for Huckabee (1961) showed that large changes in lactate production can occur without cellular hypoxia and can be related to increased pyruvate production. The concept of excess lactate was used to distinguish between pyruvate-induced changes and those related to a shift in the DPN-DPNH (diphosphopyridine nucleotide and reduced form) system. The DPN-DPNH system reflects the state of cellular oxygenation; when cellular hypoxia occurs, there is a shift toward the reduced form. The lactate-pyruvate equilibrium is useful to indicate the state of oxidation of DPN since both substances are highly diffusible and lactate is a final metabolic step. Cyanide resulted in excess lactate production in dogs aa did exercise and hypoxia (Huckabee, 1961; Krasnow et al., 1962b). It was estimated that anaerobic metabolism of the heart accounted for 11% of the total energy production during hypoxia and 12% during exercise (Huckabee, 1961). Other studies in dogs showed that excess lactate production did not occur under hypoxic conditions until the arterial oxygen saturation declined below 9 vol% (Ballinger and Vollenweider, 1962). This was accompanied by a rise in the RQ of the heart to values in excess of 1 ; the excess lactate production paralleled the rise in RQ. After 4 minutes of total anoxia (breathing 100% nitrogen) ventilation with 100% oxygen produced a resumption of aerobic metabolism with removal of excess lactate and a reduction of the RQ to below 1. These studies demonstrate that anaerobic metabolism can occur in the heart even though all of the oxygen available in the blood is not utilized. However, there is no indication of the state of oxygenation a t the cellular level. Another approach to estimate the state of oxidation of the DPNDPNH system is the estimation of oxidation-reduction potential (redox) of the lactate-pyruvate system in cardiac muscle cells (Gudbjarnason et al., 1962; Stock et al., 1962). When the cellular oxygen supply is inadequate for metabolic needs the oxidation-reduction systems approach a more reduced state and the redox potential (Eh) decreases. Direct determination of the redox potential of the intact heart is not possible. However, the calculated lactate-pyruvate redox potentials in the arterial and coronary sinus blood bear a relationship to that in heart muscle. In dogs with adequate oxygenation the heart muscle and coronary sinus had a more positive redox potential than arterial blood (AEh positive). In the

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anoxic heart the redox potential of the heart and sinus blood was more negative than the arterial blood (AEh negative). A positive AEh indicates active oxidation and that the energy is supplied by oxidative phosphorylation. A negative AEh indicates glycolysis and that part of the energy comes from anaerobic phosphorylation. In patients with uncomplicated coronary disease AEh was positive a t rest and became more positive on exercise, indicating no over-all deficiency in aerobic metabolism. These results confirm those previously discussed with the excess lactate method. During anoxia of heart muscle, regardless of the mechanism, there was rapid disappearance of glycogen (glycogenolysis) and an increase in lactate and hexose monophosphate (Conn et al., 1959; Bing et al., 1960; Danforth et al., 1960; Bing, 1961). The rise in lactate and hexose monophosphate accounted for the decline in glycogen (Conn et al., 1959; Danforth et al., 1960; Bing, 1961). The levels of fructose lJ6-diphosphate, dehydroxyacetone, or pyruvate were unchanged (Danforth et al., 1960) or decreased (Bing et al., 1960). This suggests that phosphofructokinase waB rate-limiting in this situation. I n addition, adenosine triphosphate (ATP) and creatine phosphate disappeared rapidly and nucleotides such as D P N and T P N decreased (Bing et al., 1960; Danforth et al.,1960). I n the intact dog heart, reperfusion with oxygenated blood, after brief periods of anoxia, permitted resynthesis of glycogen and ATP (Bing, 1961). After 15 minutes of anoxia, restoration of aerobic conditions did not result in effective contractile activity ; hexose monophosphate declined to normal, ATP rose for a short time, but the decline in glycogen continued (Danforth et al., 1960). This would indicate damage to the enzymic system leading to glycogenesis a t least. I n special studies using intact dogs the heart was arrested with potassium chloride and coronary perfusion was interrupted for 0.5 to 5 hours after which the heart was reperfused with oxygenated blood (Bing et al., 1958; Michal et al., 1958). Oxygen consumption and carbohydrate metabolism were determined before and after the period of circulatory arrest. Oxygen consumption was normal after 2 hours of circulatory arrest but after this there was a rapid decline. After 4 hours of ischemia, oxygen consumption was only 25% of the preischemia control value (nonbeating) . There were severe disturbances of carbohydrate metabolism indicative of aerobic glycolysis (oxygen uptake declined more than glucose uptake). No doubt if the heart had been beating in these studies there would have been a more rapid failure of the oxidative enzyme systems. I n isolated papillary muscles which were driven electrically, anoxia (nitrogen) resulted in a rapid decline in contractile activity in the presence or absence of glucose (Winbury, 1956). Reintroduction of oxygen after 60 minutes of anoxia permitted little recovery of contractile activity

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when glucose was absent during the anoxic period. However, when glucose was present during the entire 60 minutes of anoxia, contractile activity returned when aerobic conditions were restored. Thus glycolysis did not produce sufficient energy to support contractile activity for prolonged periods but did permit maintenance of the enzymic pathways and the contractile mechanism during anoxia. This may be related to the observation that pretreatment of dogs with glucose 2 hours before removing the heart resulted in higher glycogen levels at zero time and after 2 hours compared with control animals (Conn et al., 1959). Ill. Progress in the Treatment of Coronary Insufficiency

Many compounds have been advocated for the treatment of angina but few of these have survived a carefully controlled clinical trial. Each year sees the development of new agents in the laboratory, but little has been added to our armamentarium for the treatment of coronary insufficiency other than modified nitrites. Theoretically the pain of angina pectoris can be relieved anywhere between the heart and the central nervous system where the pain is finally perceived. Reduction in pain or increasing coronary blood flow are the two main approaches that have been used for treatment in the past (Katz, 1956). However, a recently developed P-adrenergic-blocking agent has been used with some success (Dornhorst and Robinson, 1962). Considering all of the factors, drugs could act on any of the following sites : 1. Coronary vessels a. Relief of spasm? b. Arteriolar dilatation c. Improvement of nutritional blood flow 2. Myocardium a. Reduction of metabolic requirement or improved metabolic efficiency b. Reduction in contractile effort c. Reduction in diastolic size 3. Arterial pressure Reduction of work load on heart 4. Heart rate Reduction of excessive rate decreases myocardial oxygen consumption 5. Effect m nervous system a. Interruption of nerve pathway for pain transmission b. Reduction of central awareness of pain

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Throughout the years a multitude of coronary dilators have been developed (Charlier, 1961) but few have stood the test of time. The basic mechanism of the coronary dilatation is of primary importance (see Section 11,B) and it is conceivable that coronary dilatation may not improve myocardial oxygenation. Gregg and Sabiston (1956) distinguished between “benign” and “malignant” coronary dilatation. A benign vasodilator should affect the coronary vessels directly and not myocardial metabolism, thereby resulting in an improved supply/demand ratio and an improved myocardial oxygenation. This presupposes that there is also improvement in nutritional blood flow which may not always be the case (Winbury et al., 1962a). A malignant vasodilator is one that has a primary effect on myocardial oxygen requirement and the increase in coronary blood flow is secondary to this. Under these circumstances myocardial oxygenation may not be improved. Implied in the discussion of coronary dilator action is the ability of the coronary bed to dilate. Certainly the results of Gorlin et al. (1959a), which suggest that nitroglycerin does not increase the coronary flow in the anginal patient, should cause one to reflect about the importance of coronary dilatation. A shifting of blood from nonnutritional to nutritional channels is a possibility and does not require any increase in the total rate of flow (Winbury et al., 1962a). The evidence for the possibility of spasm of the coronary is equivocal and, as indicated in Section I, spasm is unlikely to be a cause of angina. This leads to the conclusion that effective drugs such as the nitrites may act by one or more of the mechanisms in 2, 3, or 4 above. Details of the pharmacology of the nitrites will be discussed in Section IV.

A. THYROID INHIBITION This form of therapy has been successful in intractable angina, presumably because of a reduction in cardiac oxygen requirement. During hyperthyroid states there is a generalized increase in tissue metabolism including the myocardium. Coronary blood flow, heart rate, cardiac output, cardiac work, cardiac oxygen consumption, and TTI (Gorlin, 196213) are elevated in thyrotoxicosis (Rowe et al., 1956). therapy reduces these parameters and thereby reestablishes an adequate coronary reserve (Rowe et aZ., 1956). It has been suggested that uncoupling of oxidative phosphorylation may occur in hyperthyroid states but a review on this subject raises questions about the presence of such a biochemical lesion (Olson, 1962b). Suppression of thyroid function has been of some success in the treatment of angina even in euthyroid patients. Initially thyroidectomy was employed (Blumgart et al., 1933) ; a t a later time thyroid-suppressing

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agents, such as thiouracil, were used (Raab, 1945), and finally IlS1became available (Blumgart et d.,1951). The effects of IlS1are particularly dramatic in the management of patients with incapacitating angina with pain a t rest or with minimal exertion (Shelburne et al., 1962). Using an experimental model of coronary insufficiency in atherosclerotic rabbits it was found that chronic thiouracil treatment reduces the severity of the ST segment depression induced by hypoxia (Tabachnick et al., 1961). B. MONOAMINE OXIDASEINHIBITORS Various monoamine oxidase inhibitors have been shown to have palliative effect on the pain of angina without any improvement in the electrocardiogram (Cesarman, 1959; Cossio, 1959; Master and Donoso, 1959; Schweieer, 1959; Grant, 1963). The exact mechanism is unknown but i t is unlikely that there is improvement in the basic coronary insufficiency since the stress electrocardiogram is still abnormal after relief of pain (Master and Donoso, 1959; Schweieer, 1959). It has been concluded that the relief of pain may be a result of mood elevation and of increased pain threshold (Master and Donoso, 1959; Grant, 1963). I n view of this, these agents must be used with caution and overexertion be avoided since the anginal pain, which is indicative of coronary insufficiency, is obtunded (Grant, 1963). A recent study by Goldberg and co-workers (1962) has demonstrated that isocarboxazid reduced the cardiovascular response to exercise. Three patients were studied and the most prominent effect was an attenuation of the rise of blood pressure, heart rate, and cardiac output normally produced by exercise. I n two of the patients angina, which appeared during a placebo period, disappeared on drug therapy. The authors suggest that the beneficial effect may be due to the attenuation of the cardiovascular response to exercise. Such an effect should be beneficial in the patient with coronary insufficiency but objective electrocardiographic evidence for a beneficial effect is lacking. The pharmacological and cardiovascular actions of monoamine oxidase inhibitors have been explored extensively in animals in an attempt to explain the palliative action in angina. Only a few reports will be discussed. Iproniazid administration for 4 days markedly reduced the incidence of fatal ventricular fibrillation produced by coronary occlusion in unanesthetized dogs (Regelson et al., 1959). This effect was not specific since pentobarbital anesthesia, morphine, reserpine, hexamethonium, and chlorpromaeine were also effective. Perfused isolated rabbit hearts were capable of beating for longer periods under anoxic conditions when iproniazid was added to the perfusion medium or when the animals received drug for 7 days prior to the experiment (Setnikar and Ravasi, 1980).

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Pretreatment of rats with nialamide, iproniazid, isocarboxazid, or pivalybenzhydrazine reduced the severity of myocardial necrosis resulting from high doses of isoproterenol (Cahn and Herold, 1962; Zbinden, 1962). I n addition, isocarboxazid reduced the severity of the electrocardiographic changes produced by intravenous injection of vasopressin in the rat (Cahn and Herold, 1962). Other studies have indicated that iproniazid and pheniprazine are ineffective in preventing the ST segment depression induced by hypoxia in atherosclerotic rabbits (Tabachnick et al., 1961; Varma and Melville, 1962a). Furthermore, iproniazid did not prevent the ST segment depression resulting from coronary ligation in dogs or injection of picrotoxin into the lateral ventricle of rabbits (Varma and Melville, 1962a). There are conflicting reports as to the coronary dilator activity of monamine oxidase inhibitors (Charlier, 1961) ; however, in the intact animal little change in coronary flow is usually the case (Bing, 1959; Gorlin, 1962b). Since monoamine oxidase inhibitors will prevent the destruction of serotonin, which is a benign coronary vasodilator (Crumpton et al., 1959), the possibility of an indirect action might be considered. At the present time there is little evidence to support this view (Calesnick, 1963), and we cannot overlook the clinical results which fail to show an improvement in the basic coronary insufficiency. C. ADRENERGIC-BLOCKING AGENTS The fact that the catecholamines produce a marked increase in cardiac oxygen requirements has been discussed in Sections 11, C, and D. Part of the increase can be attributed to hemodynamic factors, such as increased vigor of contraction, producing a rise in the TTI, and part is due to a direct effect on myocardial metabolism, increasing oxygen consumption over and above that required for the additional work. With a limited coronary reserve as found in coronary insufficiency it is easy to understand why an increase in cardiac oxygen requirement will result in an imbalance between the supply and demand. Raab (1956, 1962) has stressed the importance of the catecholamines in angina but it was only recently that there was clinical evidence to support this hypothesis. In a carefully controlled clinical study Dornhorst and Robinson ( 1962) found that the selective 8-adrenergic-blocking agent, nethalide, increased the exercise tolerance of anginal patientsl; there was an attenuation of the tachycardia due to exercise but the increase in cardiac output or stroke volume was not altered. Studies in animals demonstrated that nethalide blocked or attenuated the positive inotropic or chronotropic effect of cardiac sympathetic stimulation, isoproterenol, or epinephrine; there was blockade of other &actions of the catecholamines

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but no effect on the a-actions (Black and Stephenson, 1962). Nethalide itself has only slight pharmacological effects, i.e., slight bradycardia in animals and man, and a slight myocardial depressant action in animals (Black and Stephenson, 1962 ; Dornhorst and Robinson, 1962). Dichloroisoproterenol (DCI) is another p-adrenergic-blocking agent that selectively blocks the positive inotropic and chronotropic actions, and the increased cardiac oxygen consumption produced by epinephrine, norepinephrine, and isoproterenol, and by cardiac sympathetic stimulation (Moran and Perkins, 1958, 1961; Hashimoto e t al., 1960; Nickerson and Chan, 1961). a-Adrenergic-blocking agents, such as dibenamine phenoxybenzamine, phentolamine, azapetine, or hydergine, did not cause a specific blockade (Hashimoto et d.,1960; Moran and Perkins, 1961; Nickerson and Chan, 1961) of these cardiac effects. D C I produces a prolonged positive inotropic and chronotropic effect which would prevent use in coronary insufficiency (Moran and Perkins, 1958).

D. CORONARY DILATORS OTHERTHANNITRITES A recent compilation of the pharmacological and clinical action of a score of coronary dilators has been prepared by Charlier (1961). Among the most recently developed compounds is dipyridamole, which has been studied extensively in both animals and man. There is no question that dipyridamole is a potent long-acting coronary dilator which increases coronary sinus oxygen saturation in dogs (Bretschneider et al., 1959; Grabner et al., 1959; Hockerts and Bogelmann, 1959; Kadatz, 1959; Jackson, 1961; Soloff e t al., 1962; West et al., 1962a). Changes in cardiac output and work were minimal ; likewise cardiac oxygen consumption and efficiency were not altered (Bretschneider e t al., 1959; Grabner et al., 1959; Kadatz, 1959; West et al., 1962a). On the basis of these findings, dipyridamole can be considered as a benign coronary dilator and should be of value in the treatment of coronary insufficiency. Clinical results in treatment of coronary insufficiency are disappointing, with the more recent studies showing little or no beneficial effect using pain, exercise tolerance, or electrocardiographic stress tests as the end point (Peel e t al., 1961; McGregor, 1962; Soloff e t al., 1962; DeGraff and Lyon, 1963). These studies utilized both the intravenous and oral routes so that poor absorption would not seem to be a factor. Yet coronary sinus catheterization studies in man demonstrated an increase in coronary blood flow and a rise in coronary sinus oxygen content after intravenous injection (Peel et al., 1961; Wendt e t al., 1962). I n fact, Wendt e t al. (1962) suggested that the action of dipyridamole on the coronary circulation of normal man is similar to that of nitroglycerin, namely, increased coronary blood flow, increased cardiac oxygen consumption, and decreased effi-

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ciency (Brachfeld et al., 1959). However, there is one difference, namely, that nitroglycerin consistently reduced cardiac work while the effect of dipyridamole was variable. These data raise the question about the importance of coronary dilatation per se in the treatment of coronary insufficiency even though the dilatation is of a benign nature. Other evidence would also suggest that dipyridamole should have a beneficial effect in coronary insufficiency. Blood flow was increased in dogs with infarcted hearts (Kiese et al., 1960) or with the lumen fixed in size (West et al., 1962a). Further there was a more rapid development of collateral circulation during gradual coronary occlusion in the dog when the animals were chronically treated with dipyridamole (Asada et al., 1962; Vineberg et al., 1962). Using the pitressin stress test in rabbits i t was found that dipyridamole reduced the electrocardiographic abnormalities normally observed (Mutti and Chiari, 1961). Also there was a reduction in the ST segment depression induced by injection of picrotoxin into the lateral ventricle of the rabbit (Varma and Melville, 1962a). Biochemical studies in animals suggest that dipyridamole may have some effect on nucleotide metabolism. The decrease in myocardial ATP levels induced by hypoxia was partially reversed by dipyridamole in intact dogs (Hockerts and Bogelmann, 1959). Using isolated atria it was observed that dipyridamole would permit recovery of the ATP and creatine phosphate levels and a return of contractile activity after digitalis arrest (Siess, 1962). In addition, when arrest was induced by hypoxia or sodium fluoride, dipyridamole caused recovery of the ATP levels (Siess, 1962). Hearts removed from rats pretreated with dipyridamole and subjected to 45 minutes of ischemia showed only an increased concentration of adenosine with no change in the total nucleotide or nucleoside content (Gerlach and Deuticke, 1963). This was probably a result of reduced breakdown of adenosine rather than increased production. It was suggested that the accumulation of adenosine may account for the coronary dilator action. This speculation is interesting in view of the recent hypothesis of Berne (1963) that adenosine is involved in the autoregulation of the coronary circulation.

E. MISCELLANEOUS AGENTS The importance of hypocholesterolemic agents in the treatment of coronary insufficiency is still questionable and many years will be required for a definitive answer. One of the agents which blocks cholesterol synthesis, triparanol, has been reported to improve coronary insufficiency in several patients after only a short period of administration (Hollander et al., 1960). This was accompanied by only minor changes in serum cholesterol so that i t seems unlikely that this improvement was associated

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M.

WINBURY

with an effect on cholesterol metabolism. Another group studied the effects of triparanol on the stress electrocardiogram in cholesterol-fed atherosclerotic rabbits (Tabachnick et al., 1961) ; chronic treatment for 1 to 2 weeks resulted in a marked reduction in the severity of the hypoxia-induced ST segment depression, After withdrawal of the triparanol the ST segment depression gradually reverted to the pretreatment value. This study suggests that triparanol may have an effect on myocardial metabolism since the coronary vessels of these rabbits were severely sclerosed. Reserpine was tested in a series of anginal patients who responded well to nitroglycerin. There was no difference between reserpine and placebo in the daily nitroglycerin requirement or exercise tolerance even though many of the patients had a lower blood pressure and heart rate while on reserpine treatment (Rosenberg and Malach, 1961). Chronic reserpine treatment of atherosclerotic rabbits intensified, rather than reduced, the electrocardiographic response to hypoxemia and produced little change in the hypercholesterolemia (Melville and Varma, 1962). IV. Action of Nitrites

The term “nitrites” may be considered a generic one referring to both nitrites and nitrates. There is still the question as to whether or not organic nitrates must be reduced to the nitrite form in the body for activity. For simplicity in this section, the term nitrite will be used to refer to all organic nitrites or nitrates that have been used for the treatment of coronary disease. These are the most useful drugs for the treatment of coronary insufficiency, with nitroglycerin somewhat of a standard because of its long successful use. There is no question that if the nitrite is absorbed i t will effectively relieve pain and improve the stress electrocardiogram. As Rineler (1962) has said, “Available evidence leads us to conclude that a nitrite is a nitrite is a nitrite.” Although nitroglycerin and other organic nitrites are the most effective agents for the relief of angina, they are not the most effective coronary dilator agents; the properties of the nitrites will be discussed in an attempt to evaluate the possible mechanisms of action. The nitrites are general smooth muscle relaxants acting on the smooth muscle of blood vessels (arteries, arterioles, and veins) and other organ systems. This action is direct (musculotropic) and not dependent on blockade of nerve pathways.

A. CORONARY HEMODYNAMIC ACTION The coronary dilator action of nitroglycerin has been demonstrated by measuring the blood flow rate in both normal animals and normal man. The increase in blood flow was not associated with an increased perfusion

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pressure indicating a reduction in coronary vascular resistance (reviewed by Charlier, 1961). Using angiocardiographic techniques in the intact dog, nitroglycerin and amyl nitrite increased the diameter of the large coronary vessels, and the small vessels were better visualized (Haight et al., 1962). Similar findings were made in anginal patients after nitroglycerin or erythrityl tetranitrate, using oral or parenteral therapy (Likoff et al., 1962). Because the smaller vessels could not be visualized, the authora concluded that one cannot imply that the increase in the caliber of the large arteries was accompanied by an improvement in coronary blood flow. Another group fortuitously studied a patient during an anginal attack and observed poor filling of the right and left coronary arteries; after isosorbide dinitrate there was relief of the angina and good filling of the right coronary artery but the left was still poorly visualized (Gensini et al., 1962). This was interpreted to indicate a spasm during the attack and its relief by the nitrite. When vasopressin was injected into the left coronary artery of the intact dog the radiopaque material did not penetrate the affected artery, indicating vasoconstriction; this was relieved by isosorbide dinitrate (Likoff et al., 1962). Studies in dogs indicate that nitroglycerin is a benign coronary dilator but this is not the case in man. I n the open-chest dog with the hemodynamic parameters permitted to vary ad Zib., injection of nitroglycerin into the coronary artery produced an increase in coronary flow accompanied by an increase in coronary sinus oxygen content, a decrease in A-V O,, and a slight increase in oxygen consumption (Eckstein et al., 1951). When the hemodynamic parameters were controlled, and cardiac work was unchanged, there was no increase in oxygen consumption (Sarnoff et al., 1958b). Others, using a fixed rate of inflow to the coronary artery, found that intracoronary injection of nitroglycerin or pentaerythritol tetranitrate reduced myocardial oxygen consumption as well as vascular resistance, but nutritional blood flow (Rbae clearance; see Section V, 1) was increased (Winbury et al., 1962a). Finally, after intravenous injection of nitroglycerin in the open-chest dog, the cardiac oxygen consumption was invariably reduced, presumably as a result of a reduction in the hemodynamic work load (blood pressure) ; changes in coronary blood flow and A-V O2were variable depending upon the degree of hypotension (Fig. 2) (Winbury and Rubin, 1961). I n normal man both coronary blood flow and cardiac oxygen consumption increased, cardiac efficiency decreased, coronary vascular resistance decreased, and the A-V 0, was unchanged after sublingual nitroglycerin (Fig. 1) (Brachfeld et al., 1959; Gorlin, 1962b). These investigators suggest that the increase in oxygen consumption may be due to uncoupling of oxidative phosphoryla-

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tion; however, one could interpret these data as indicating the existence of an oxygen debt in the normal heart and when more blood was supplied the debt was temporarily satisfied. The experimental evidence in support of either thesis is weak a t the present time (McKeever e t al., 1958; Honig et al., 1960). In the anginal patient, coronary blood flow decreased to the same extent as blood pressure, and coronary resistance was unchanged

FIQ.2. Effect of nitroglycerin (2.5 pg/kg) on coronary circulation and cardiac oxygen consumption. Nitroglycerin was given intravenously; point of injection indicated by vertical line. CBF: coronary blood flow (ml/min); A SAT and CS SAT: arterial and coronary sinus oxygen saturation ( ~ 0 1 %;) BP: blood pressure (mm Hg) : upper line systolic, lower line diastolic; O2CONS: cardiac oxygen consumption (ml/min) . Note decrease in oxygen consumption.

(Fig. 1) (Gorlin et al., 1959a; Gorlin, 1962b). Erythrityl tetranitrate did not alter coronary blood flow or cardiac oxygen consumption in the normal or anginal patient ; however, coronary vascular resistance did decline (Rowe et al., 1961). The reason for the discrepancy between these results and those previously discussed (Brachfeld et al., 1959; Gorlin et al., 1959a; Gorlin, 196213) is difficult to explain. ACTIONS B. GENERALHEMODYNAMIC In recent years many have suggested that the beneficial effect of the nitrites in the relief of angina is extracoronary and could well be associated with a reduction in the work load and oxygen requirement of the heart (Gorlin et al,, 1959a,b; Darby and Aldinger, 1960; Rowe et al., 1961; Darby and Gebel, 1962; Gorlin, 1962b; McGregor, 1962; Calesnick,

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1963). If one were to consider the number of agents that are far more effective coronary dilators than nitroglycerin but are ineffective in the treatment of angina, the above conclusion is inevitable. Further, the evidence of Gorlin et al. (1959a,b) suggests that nitroglycerin does not increase the coronary blood flow in the anginal patient; Rowe found the same to be true for erythrityl tetranitrate (Rowe et al., 1961). Some of the hemodynamic alterations produced by nitroglycerin (and other organic nitrites) are decrease in blood pressure (systolic more than diastolic) (Brachfeld et al., 1959; Gorlin e t al., 1959a; Rowe et al.,1961; Gorlin, 1962b) ; decrease in right and left atrial pressure (Brachfeld et al., 1959; Gorlin et al., 1959a; Rowe et al., 1961; Gorlin, 1962b) ; decrease in cardiac size (Darby and Aldinger, 1960; Darby and Gebel, 1962; Gorlin, 1962b) ; decrease in left ventricular work (Brachfeld et al., 1959; Gorlin et al., 1959a; Rowe et al., 1961; Gorlin, 1962b) ; decline in TTI (Gorlin, 1962b) ; increase in heart rate (Brachfeld et al., 1959; Rowe et al., 1961), but decline in systolic duration (Gorlin, 1962b) and reduction in the rise in blood pressure and pulmonary arterial pressure during exercise (Gorlin, 1962b; Stock et al., 1962). Cardiac output was either unchanged (Brachfeld et al., 1959) or reduced (Gorlin et al., 1959a; Rowe et al., 1961). Many of these changes are interrelated and will reduce TTI which, as indicated in Section 11, C, is the major determinant of cardiac oxygen consumption. Certainly the decrease in blood pressure, diastolic heart size, and systolic duration will decrease TTI and the wall tension of the heart. The classic reports of Brachfeld e t al. (1959) and of Gorlin et al. (1959a, 1962b), which show that nitroglycerin increased coronary blood flow and cardiac oxygen consumption in normal individuals but reduced both parameters in the anginal patient, clearly demonstrated the factors leading to the reduction in cardiac work and TTI. Although the general hemodynamic changes were similar on a qualitative basis in both groups, there are quantitative differences. There was a greater decrease in blood pressure, cardiac output, cardiac work, and TTI, and a smaller rise in heart rate in the anginal patient (Fig. 1).The decline in cardiac efficiency was considerably less in the anginal than normal individual. It is paradoxic that in spite of the decrease in cardiac work and wall tension, oxygen consumption increased or remained unchanged. I n the dog, nitroglycerin given intravenously has positive inotropic and positive chronotropic effects which are reflex in origin, since the effects can be blocked by spinal anesthesia (Darby et al., 1958).Administration of nitroglycerin during infusion of norepinephrine caused a reduction in end diastolic tension and length, presumably as a result of the decline in blood pressure (Darby and Aldinger, 1960). During an anginal attack induced by exercise there was increased heart size which could be reduced

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by nitroglycerin (Darby and Gebel, 1962). This, of course, results in a reduction in wall tension and cardiac oxygen requirement. Nitroglycerin produces an “unsteady state” in the circulation of normal dog and man with an initial increase in cardiac work for about 1 minute followed by a decline (Honig e t al., 1960; PoijB and Rudewald, 1962). Kinetic work (flow) accounted for almost all of the increase; pressure-volume work was relatively unchanged (Honig e t al., 1960). The authors explained the increased cardiac oxygen consumption produced by nitroglycerin in normal man on the basis of the increased cardiac work, but this does not follow, since kinetic work involves very little tension development and therefore requires little additional oxygen consumption. I n these studies (Honig e t al., 1960; Poij6 and Rudewald, 1962) there was an over-all decline in blood pressure a t the time of the increased cardiac output, which should result in a reduction of TTI. Nitroglycerin decreases the degree of rise in blood pressure, cardiac work, and TTI produced by exercise (Gorlin, 1962b). Further, i t can prevent the rise in pulmonary arterial pressure, left atrial pressure, and cardiac size that may occur in anginal patients on exercise (Darby and Gebel, 1962; Stock et al., 1962). It has been concluded that there may be left ventricular failure during angina of effort which is relieved by nitroglycerin. This beneficial effect is probably associated with the reduction in the hemodynamic work load on the heart reducing the wall tension.

C. CARDIAC METABOLISM A comprehensive study of the effects of nitroglycerin on myocardial metabolism in intact dogs by Goto (1962) indicated a general decline in cardiac metabolism with a shift toward carbohydrate utilization. Intramuscular administration of 0.5 gm/kg produced a marked decline in the utilization of oxygen, NEFA, and oleic acid but total carbohydrate utilization was only slightly reduced. Glucose and pyruvate utilization declined, but an increase in lactate utilization compensated for most of the decline. The slight rise in RQ (0.81 to 0.89) agrees with the slight shift toward carbohydrate metabolism. The general decline in metabolism was associated with a decline in arterial pressure but the studies do not permit one to determine how much of the general decline in cardiac metabolism was due to the reduced cardiac work load. A preliminary report by other investigators (McGregor, 1962) suggests that nitroglycerin does influence the uptake of substrates by the myocardium and that this may have some bearing on the manner in which i t relieves angina pectoris. The apparent “oxygen wasting” in normal man is difficult to understand. This has been explained by an uncoupling of phosphorylation and an inhibition of electron transport produced by nitroglycerin in in vitro

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studies (Brachfeld et al., 1959; Honig et al., 1960; Calesnick, 1963). Use of in vitro metabolic data for explanation of an in vivo result is not without pitfalls and a t present it would be best to assume that the oxygen wasting in normal man is unexplained. Organic nitrites and nitrates inhibit adenosine triphosphatme activity in arterial smooth muscle and it was suggested that this may be related to the vasodilator action (Krantz et al., 1951).

D. EFFECT ON CATECHOLAMINES Raab (1956) has stressed the importance of the catecholamines and the sympathetic nervous system in the precipitation of coronary insufficiency. Indeed, during exercise, plasma levels of norepinephrine and epinephrine increased significantly (Gazes et al., 1959; Chidsey et al., 1962). Raab and Lepeschkin (1950) suggested that nitroglycerin has an antiadrenergic effect on the heart and blocks the wasteful increase in oxygen consumption produced by endogenous release of catecholamines. The studies on which this conclusion was based were carried out in atropinized cats (Raab and Lepeschkin, 1950). The tachycardia and T wave depression which follow administration of epinephrine or norepinephrine were blocked by nitroglycerin. Popovich et al. (1956) were unable to confirm these results even though the same procedures and animal species (cat) were used. In addition, nitroglycerin did not prevent the increase in myocardial oxygen consumption induced by catecholamines or cardiac sympathetic nerve stimulation in the cat or dog (Eckstein et al., 1951; Popovich et al., 1956; Winbury et al., 1962a). Likewise, nitroglycerin had no apparent effect on the increase in isometric systolic tension produced by norepinephrine (Darby and Aldinger, 1960). The evidence to the present does not support the thesis that nitroglycerin has a true antiadrenergic effect. It is true that it can counteract the increased hemodynarnic work load due to norepinephrine (Darby and Aldinger, 1960), but this is merely due to an opposing physiological action on blood pressure.

E. COMPARISON OF RESPONSE IN NORMAL AND ANGINAL INDIVIDUAL There is a marked difference in the response of the coronary circulation

to nitroglycerin in the normal human and the anginal patient (Fig. 1).I n the normal, there was a rise in coronary blood flow, a decline in coronary vascular resistance, and an increase in left ventricular oxygen consumption (Brachfeld et al., 1959; Gorlin, 1962a). On the other hand, in the anginal patient coronary flow declined, coronary resistance was unchanged, and cardiac oxygen consumption declined (Gorlin et ul.,1959a,b; Gorlin, 1962b). Other investigators found that erythrityl tetranitrate pro-

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duced no change in coronary flow or cardiac oxygen consumption in normal or anginal patients, but did decrease coronary resistance (Rowe et al., 1961). The clearance of 1131 in anginal patients following percutaneous injection into the left ventricular apex was not altered by nitroglycerin (Hollander et al., 1963), confirming other reports that coronary flow was not increased (Gorlin e t al., 1959a,b; Rowe et al., 1961; Gorlin, 196213). It can be inferred, from these observations, that the coronary blood flow rate is relatively fixed in the anginal patient (Rowe, 1962). Nitroglycerin was studied in an animal model of coronary insufficiency, the atherosclerotic rabbit, and did not prevent the S T segment depression induced by hypoxia (Winbury et al., 1961; Varma and Melville, 1962a). When isolated perfused rabbit hearts were studied, i t was found that nitroglycerin produced a greater increase in coronary blood flow in normal hearts compared with atherosclerotic (‘coronary insufficient hearts” (Karp et al., 1960; Melville and Varma, 1962). This animal model demonstrates an impaired ability of the atherosclerotic coronary vessels for dilatation.

F. EFFECT ON COLLATERAL CIRCULATION Gradual occlusion of the right coronary artery of the pig produced death in 13 of 15 pigs, Treatment of animals with pentaerythratol tetranitrate before and during the occlusion increased the survival rate significantly (Lumb e t al., 1962; Lumb and Hardy, 1963). Filling of the coronary circulation with a radiopaque mass demonstrated an extensive collateral circulation in the treated animals. A similar improvement in the collateral circulation has been reported for sodium nitrate (Zoll and Norman, 1952). G. CONCLUSION

It appears that the coronary bed of the anginal patient cannot increase the volume flow under the influence of nitrites, Therefore the beneficial effect must be associated with some other change such as a reduction in wall tension of the heart. It is conceivable that there may be a redistribution of blood with more blood going to nutritional vessels and permitting better tissue oxygenation in that way. V. Approaches to laboratory Evaluation of Antianginal Agents

The coronary circulation is a complex integrated system involving many variables which were emphasized in Section 11. An analysis of the influence of drugs on the coronary circulation, of necessity, should consider these variables in order to have a rational basis for predicting possible utility in coronary insufficiency (Katz, 1956). However, the majority of physiological and pharmacological studies have been carried out in

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animals with a normal coronary circulation and the results obtained may not predict the status of the abnormal coronary circulation. Certainly studies in man indicate that the coronary bed of the anginal patient is incapable of dilatation so that nitroglycerin produces no increase in coronary flow (Gorlin et al., 1959a,b; Gorlin, 1962b). Yet, the pharmacologist has been using coronary dilatation in the normal animal as the basis for the evaluation of drugs for coronary insufficiency (Charlier, 1961). It is possible that dilatation may play some part in the action of the nitrites but there are other actions involved, as was mentioned in Section IV. Where does the reduction in the work load of the heart fit in and how can we evaluate agents on a more logical basis? If we could have an animal replicate or procedure that takes into account the abnormal physiology, we would be on firmer ground and perhaps could increase the probability of discovering new agents or approaches to the treatment of angina. Many approaches have been used to investigate the physiology and pharmacology of the coronary circulation and over-all metabolic function of the heart. Some of these have not yet been applied to the study of new drugs and it is the purpose of this section to consider the approaches that can be drawn from the fields of physiology, pharmacology, and biochemistry, Among the topics that will be discussed are: coronary dilatation; total metabolic studies ; measurement of myocardial oxygen tension ; various attempts a t producing experimental coronary insufficiency ; prolongation of activity under hypoxia ; antagonism of catecholamines ; arteriographic techniques ; and coronary capillary circulation.

A. CORONARY DILATOR ACTION The ability of agents to dilate coronary arteries has been determined in a variety of ways, ranging from isolated strips or segments to determination of the rate of coronary blood flow or the caliber of the coronary arteries in situ (Kranta and Ling, 1958; Charlier, 1961). These procedures have all been based on the fact that nitroglycerin is a good coronary dilator and is the drug of choice for the relief of angina (McGregor, 1962). Isolated arterial strips or segments are useful as a tool for understanding the pharmacology of a compound, but because of the complexity of the integrated coronary circulation it would be difficult to predict what can be expected in the intact animal. Furthermore, there is variation among the species in the response to various agents. I n addition, the larger vessels studied may not reflect the response of the arterioles. 1. Isolated Heart The isolated heart procedure originally described by Langendorff (1895) is probably the most frequently used method for evaluating coro-

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nary dilator action. It has the advantage over isolated arterial rings or segments of permitting simultaneous evaluation of the effect of drugs on the coronary circulation and cardiac action. Although there have been many diverse technical modifications of the procedure, the basic principle of the preparation remains unchanged. The coronary arteries are perfused at a constant pressure via the aorta with a balanced salt solution (or blood) and the rate of coronary blood flow is determined as the outflow from the right heart, Drugs under study are added to the perfusion fluid. I n order to study the vasomotor effects on the coronary bed independent of the extravascular component, some have induced fibrillation of the heart (Katz et d.,1938). Although the isolated perfused heart has certain advantages over isolated rings or segments because of the intact coronary bed, it does not provide information on the over-all effect that can be anticipated in the intact animal. Certainly the predictability of what effect might be expected on the coronary circulation in man can be better obtained from the intact animal, where the heart is performing work and the coronary circulation is influenced by the numerous hemodynamic variables that are normally in operation. To my mind the intact animal would better serve for the initial evaluation of drugs, and if one then wants to explore the mechanism of the effect on the coronary circulation, the isolated heart can then be used. It is true that there is a significant correlation between the potencies of a series of compounds determined by the Langendorff procedure and those obtained in the intact dog by intracoronary injection (Winbury et al., 1950). However, the correlation is low, and there are many changes noted in contractile activity of the isolated heart which are not observed in the intact animal. Recent work by Karp et al. (1960) and by Melville and Varma (1962) showed that the response of the isolated perfused heart from atherosclerotic rabbits differs from hearts obtained from normal animals (see Section V, E) , For example there is a qualitative difference in the response to ergonovine which invariably caused coronary constriction in the atherosclerotic heart but no change or dilatation in the normal heart (Karp et al., 1960). A quantitative difference was noted in the response to nitroglycerin with the normal heart showing greater dilatation (Karp et al., 1960; Melville and Varma, 1962). 2. Heart in Situ

It is only in the intact animal that one can determine, simultaneously, the effect of an agent on the coronary circulation and other hemodynamic parameters, Since the ultimate use of an agent will be in the human, i t is critical to assess the hemodynamic actions a t an early stage, because some factor may be undesirable. It would appear that compounds should first

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be evaluated in the intact animal in order to reduce the number of false positives and t o enable early elimination of those agents which have undesirable effects that can be observed only with an intact circulation. When a drug is injected directly into the coronary artery, the situation is similar to that of the isolated perfused heart and, in fact, there is reasonable correlation between both procedures (Winbury et al., 1950). Thus, intracoronary injection in the intact animal with direct measurement of blood flow is as useful and rapid a procedure for assaying coronary dilator action as the isolated heart (Winbury et al., 1950). The added advantage is that the heart is under a relatively intact humoral, nervous, and hemodynamic control so that autoregulation is more likely to occur. I n addition, the direct chronotropic and inotropic actions can be determined. When the drug is injected intravenously, we are closer to the clinical situation for which the compound is intended, and the hemodynamic factors that are integrated in the regulation of coronary flow are in operation. Most agents that produce vasodilatation of the coronary arteries will produce a similar effect in other areas and may lead to an excessive fall in blood pressure. It is the balance between these actions that determines whether or not the blood flow will increase after intravenous injection of a vasodilator agent. Although a moderate decline in blood pressure will in effect reduce coronary perfusion pressure, this may be beneficial because of the reduction in pressure work (see Section 11, C), providing i t is not associated with a marked increase in cardiac output or heart rate. After intravenous injection, nitroglycerin may or may not produce an increase in blood flow depending on the degree of hypotension ; however, coronary vascular resistance is reduced. On the other hand, dipyridamole invariably produces a prolonged increase in blood flow after intravenous injection even when blood pressure is reduced. In view of the fact that coronary dilator agents will produce marked vasodilatation in other vascular areas, the changes in coronary flow and femoral flow produced by intravenous injection of 8 series of agents have been determined simultaneously with rotameters. It is of interest that all the compounds, including nitroglycerin, produced a greater percentage and absolute increase in femoral flow than in coronary flow. For example, 2-diethylaminoethyl dicyclohexylcarbamate caused a coronary flow (anterior descending) increase of 29% while femoral flow increased 100% (Winbury and Hambourger, 1953). Under similar circumstances nitroglycerin produced a smaller increase in coronary flow even though there was a similar increase in femoral flow. To determine which of the compounds had more selectivity for the coronary bed, the relationship of change in coronary flow per unit change in femoral blood flow was calculated for several doses of each compound, and the two compounds which showed

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better selectivity for coronary dilatation were evaluated further (Winbury and Hambourger, 1953). These compounds did not alter stroke volume, cardiac output, heart rate, or cardiac work, and did reduce coronary vascular resistance ; but when tested in humans with coronary insufficiency, no beneficial effect was observed (Winbury and Hambourger, 1953). These studies merely serve to emphasize that coronary dilatation without other hemodynamic changes does not indicate whether or not a compound will be effective in man. It may be that the dilatation was malignant (Gregg and Sabiston, 1956; Gorlin, 1962b) but that is unlikely because cardiac work was unchanged. The fact that compounds appear to show some selectivity for the coronary vasculature rather than the peripheral vasculature proves little, since a vasodilator in one bed will generally cause dilatation in another bed and specificity is unlikely. It would appear that the use of such methods for the evaluation of antianginal activity is fraught with danger, and many false positives will result. To emphasize this point further there are the data of Charlier (1961) comparing the ratio of the coronary dilator potency to the peripheral dilator potency for a series of compounds using a common standard for both procedures. The coronary dilator activity was determined in the isolated rabbit heart, and the direct peripheral dilator activity (intra-arterial) in the femoral bed of the intact dog. The index of coronary dilator potencyJfemora1 dilator potency showed many agents superior to nitroglycerin, yet none of the agents is effective in the treatment of coronary insufficiency in man. The ratio indicates the relationship between the direct vasodilator activity for the two vascular areas but does not indicate what might be expected in the intact animal or man following intravenous administration. a. Direct measurement of blood flow. These procedures are usually carried out in anesthetized open-chest animals with the dog being the most frequently used species. The rate of blood flow in the coronary bed can be estimated by measuring the outflow from the coronary sinus or the bypassed right ventricle, or by measuring the inflow to one or more of the coronary arteries. Recently it has been possible t o monitor, continuously, the inflow in the unanesthetized normal animal by means of an electromagnetic flowmeter using a permanently implanted probe on a branch of the left coronary artery (Gregg, 1962a). The original procedure for cannulation of the coronary sinus via the atrial appendage (Morawitz and Zahn, 1912) has been subjected to some criticism because the ratio between sinus outflow and total left coronary inflow can vary under different physiological circumstances (La fontant et al., 1962). I n spite of these objections it appears that the coronary sinus flow is derived almost entirely from the left ventricle (Rayford et al., 1959; Moir et al., 1963) and can serve to indicate directional changes

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in coronary flow rate (Gregg, 1950). However, caution must be observed to avoid increasing the pressure within the coronary sinus by use of too small a cannula or by resistance imposed by a flowmeter, since blood may be diverted directly into the cardiac chambers. A recent study by West et al. (1962a) described the cannulation of the coronary sinus via the external jugular vein and recording outflow with a rotameter. A branch of the left coronary artery was catheterized via the carotid artery under fluoroscopic guidance. This procedure has the advantage of a closed thorax, permitting normal respiration and more physiological hemodynamic conditions. I n spite of these advantages, one wonders about the resistance imposed by the coronary sinus catheter and rotameter, and how these affect sinus outflow. To avoid the problems in accurately determining coronary outflow, Rodbard et al. (1953) described a procedure which permits collection of total coronary outflow in the right ventricle by bypassing systemic venous return. Considerable surgery is involved in this preparation and the right ventricle is performing little work. In many experiments, arterial pressure was a t levels well below 100 mm Hg, which suggests either reduced cardiac function or peripheral dilatation. Direct measurement of the rate of coronary inflow can be effected by a variety of flowmeters (orifice meter, rotameter, bubble flowmeter, electromagnetic flowmeter, thermostromuhr) . It is necessary to cannulate the coronary arteries either externally or internally through the aorta and coronary ostia except for the electromagnetic flowmeter or thermostromuhr which can be placed around the isolated branch of an artery. Using the rotameter for measurement of inflow, a rather reproducible coronary dilator assay was described which measured other hemodynamic variables simultaneously (Winbury et al., 1950). The recent development of small electromagnetic flowmeter probes permits the use of chronic preparations (dogs) in the unanesthetized state (Gregg, 1962a). Thoracotomy alters cardiac function so that hemodynamic changes observed while the thorax and pericardium are open may differ from those in the normal animal (Rushmer, 1961). b. Indirect measurement of blood flow.The nitrous oxide technique permits estimation of the rate of coronary blood flow without thoracotomy and has been used extensively in man and animals. The majority of investigators use the desaturation method of Goodale and Hackel (1953a). The computation of the rate of coronary blood flow is based on the Fick principle and requires measurement of nitrous oxide levels in the arterial and coronary sinus blood after the heart has reached saturation. This necessitates catheterization of the coronary sinus and a systemic artery and withdrawal of blood over a 5-minute period during desaturation of the

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heart. Blood flow is expressed as m1/100 gm left ventricle. This procedure requires no major surgical intervention and minimal anesthesia so that physiological conditions are more normal than when blood flow is measured directly in the thoracotomized animal. The time required for a determination ( 5 minutes) may be a disadvantage when a compound has a short duration of action. Nevertheless, the nitrous oxide method has been a valuable tool in the study of the coronary circulation of man and animals and the effect of drugs thereon. It is the only practical method available for studies in man. 3. Conclusicms Whenever coronary dilator action is studied in animals using either the isolated heart or the heart in situ we are investigating normal coronary arteries and are assuming that similar changes will obtain in the coronary arteries of the anginal patient. This is probably not the case and may partially explain why coronary dilator procedures have been unsuccessful in predicting clinical antianginal activity. The coronary circulation of atherosclerotic animals responds differently than that of normal animals, but further physiological and pharmacological studies are required before we can assume that the response of the atherosclerotic animal is similar to the response of the anginal patient. Another objection to the study of coronary dilator action alone is that the dilatation may be malignant in nature and does not improve the oxygen supply to the myocardium. Thus it is important to evaluate the effects on myocardial oxygen consumption simultaneously with the effects on coronary circulation. None of the blood flow methods distinguish between nutritional and nonnutritional flow so that there is no index of actual blood delivery to the capillaries. This will be considered in Section V, I. B. TOTAL METABOLIC APPROACH Flow measurements alone or even in conjunction with hemodynamic factors have limited value in the evaluation of antianginal agents unless the effect on myocardial oxygen metabolism is also studied. Any agent that increases myocardial oxygen requirement, either directly or by increasing the work of the heart, will produce coronary vasodilatation which is malignant (Gregg and Sabiston, 1956; Gorlin, 1962b) in nature. This is part of the autoregulation mechanism (Berne, 1963) previously discussed (see Section 11). 1. Arteriovenous Oxygen Difference (A-V 0,)

If the coronary arteries are capable of normal vasodilatation (adequate coronary reserve) an agent which would increase coronary blood

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flow in excess of that required would be useful even if oxygen demand were increased, Such an agent would decrease A-V 0,. I n fact, on a theoretical basis, any agent that would diminish the A-V 0, should be useful since the oxygen supply would be in excess of the demand. Such a reduction could result either from an increase in blood flow or a reduction in the demand or a combination of both. However, measuring of A-V 0, alone could result in many false positives and actually missing potentially effective agents. For example, a metabolic poison such as cyanide will prevent tissue utilization of oxygen (demand reduced) and thereby diminish A-V O2 (Neil1 et al., 1963b). Also, a n agent that would increase non-nutritional blood flow (arteriovenous shunts) would have the same effect because the venous blood in the coronary sinus would be diluted with shunted arterial blood. Although intracoronary injection of nitroglycerin diminishes A-V 0, (Sarnoff et al., 1958b; Winbury et al., 1962a), there is little or no change after intravenous injection because coronary blood flow is actually reduced (Fig. 2). A compound which reduces the A-V 0, in animals because of an increase in coronary blood flow may not have the same effect in the anginal patient since the coronary reserve is limited, I n an attempt to take this factor into account we have perfused the circumflex and descending branches of the coronary artery a t a constant rate and have recorded arterial and coronary sinus oxygen saturation continuously with an oximeter, in addition to the perfusion pressure (Winbury et d.,1962a). Thus the A-V O2 can only be influenced by changes in demand and actually reflects changes in left ventricular oxygen consumption. Using such a procedure, it was found that intracoronary injection of norepinephrine and epinephrine increased A-V 0, and nitroglycerin and pentaerythritol tetranitrate diminished A-V 0, (Winbury et al., 1962a). 2. Oxygen Consumption of the Heart in Situ

Simultaneous evaluation of changes in coronary blood flow and myocardial oxygen consumption permits a more rational analysis of the mechanisms involved in the changes in coronary flow. Such an analysis is a necessary requisite before a compound should be considered for evaluation in man (Katz, 1956; Calesnick, 1963). Myocardial oxygen consumption is computed from the arteriovenous oxygen difference times the rate of coronary flow [A-V 0, ( ~ 0 1 % )X coronary flow rate (ml/min)]. To reduce the oxygen consumption to basal terms, the weight of the area perfused by the coronary artery under study .should be known and the value expressed as cm8 O,/lOO gm.This is not important for the evaluation of drugs, since the oxygen consumption after drug is usually compared with the untreated state in the same animal.

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Most investigators have determined the oxygen content of the coronary sinus and arterial blood a t intervals before and after drug administration using conventional volumetric or manometric techniques. It is critical that the coronary flow rate used in the computations correspond temporally to the time the samples were drawn from the coronary sinus and artery. The development of the oxygen electrode and oximeter has made possible continuous recording of the oxygen tension or saturation of blood so that myocardial oxygen consumption can be determined almost continuously. It will be seen that this has certain advantages since compounds can have a biphasic effect which would not be uncovered unless continuous recording were possible. Nevertheless, measurement of myocardial oxygen consumption, in conjunction with other factors that affect coronary blood flow, has provided important information about the physiology and pharmacology of the coronary circulation in animals and man. Various experimental designs have been used in animals depending upon the objective of the study. The major differences are (1) the manner in which the coronary arteries are supplied with blood-autoperfusion at arterial pressure (Popovich e t al., 1956; Schreiner et al., 1957; Berne, 1958; Crumpton e t al., 1959; Scott and Balourdas, 1959; West et al., 1962a), or constant pressure (Eckstein et al., 1951 ; Berne, 1958), or constant flow (Winbury et al., 1962a) ; (2) cardiac action-fibrillation (Berne, 1958; Beuren e t al., 1958; McKeever e t al., 1958), arrest (Beuren et al., 1958; McKeever et al., 1958), or beating heart; (3) regulation of hemodynamic parameters (Sarnoff e t al., 1958b) (aortic pressure, heart rate, and cardiac output) or permissive variation; and (4) route of injection-intracoronary or intravenous. Blood flow can be measured by direct methods or by the nitrous oxide procedure (Crumpton e t al., 1959; Scott and Balourdas, 1959; Wendt et al., 1962). Basic to the computation of the oxygen consumption of the left ventricle is the assumption that the coronary sinus represents venous blood primarily from the area supplied by the left coronary artery (left ventricle) with only a small amount from the right coronary. A recent study in which left coronary inflow and coronary sinus outflow were measured simultaneously in the open-chest dog under different physiological circumstances demonstrated that the sinus flow was derived almost entirely from left coronary inflow (Rayford et al., 1959). These data suggest that a reasonably accurate value for left ventricular oxygen consumption can be obtained from measurement of left coronary inflow and the arteriocoronary sinus oxygen difference. Another group measured separately the flow and oxygen content in the coronary sinus and that entering the empty right ventricle using a systemic venous return bypass technique (Rodbard

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e t al., 1953). The oxygen content of the two drainage systems differed significantly but the coronary sinus represented blood draining primarily from the left ventricular musculature (Lafontant et al., 1962). By direct injection of I131-albumin into the septal branch of the left coronary artery it was estimated that 80% of the septal artery outflow drains directly into the right ventricle (Moir et al., 1963) ; this accounts for 12.8% of the total left coronary inflow. This study confirmed previous work indicating that the coronary sinus receives venous blood almost entirely from the left ventricle. If the venous blood from the septal area differs markedly from that of the area supplied by the circumflex and descending branches, an error can be introduced into the value for left ventricular oxygen consumption (Moir et al., 1963). However, if only the circumflex and descending branches are perfused directly, this potential inaccuracy can be avoided since the coronary sinus primarily drains that inflow. Intracoronary injection of agents shows the direct action on the heart but compounds may alter myocardial oxygen consumption by extracardiac mechanisms such as a reduction in aortic pressure. Nevertheless, it is of interest to determine the direct action during evaluation of a compound. Of greater importance are the effects following intravenous injection. Some of our experimental results will be used to illustrate various points. These studies were carried out in anesthetized dogs under artificial respiration. A left thoracotomy was performed and the circumflex and/or descending branch of the left coronary artery cannulated. The inflow to the artery was supplied by the carotid artery and a rotameter was interposed into the circuit to measure the blood flow rate. Blood pressure was determined from a side arm connected to the carotid artery. A small catheter was placed to the coronary sinus via the right atrium or the jugular vein and samples were drawn continuously through an oximeter a t a constant rate and returned to the animal via the contralateral jugular vein. Arterial oxygen saturation was recorded continuously in the same fashion using a bypass in the carotid or femoral artery. Oxygen consumption was computed a t 10-second intervals from the records. All the drugs were injected intravenously. Norepinephrine and epinephrine invariably increased myocardial oxygen consumption and coronary flow but did not alter A-V 0,. The experiment in Fig. 3 is of particular interest because blood pressure did not increase, yet coronary flow and oxygen consumption were elevated. Angiotensin produced a marked and prolonged increase in oxygen consumption, coronary blood flow, and blood pressure; the A-V 0, was diminished (Fig. 4). This illustrates the problems of using A-V 0, alone. Histamine and acetylcholine showed a biphasic effect on oxygen consump-

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MARTIN M. WINBURY

FIG.3. Effect of norepinephrine ( 1 pglkg) on coronary circulation and cardiac oxygen consumption. Norepinephrine was given intravenously. Note increase in cardiac oxygen consumption. Remainder of legend as in Fig. 2.

tion which might not have been observed if continuous recording of A-V 0, were not possible (Fig. 5 ) . The elevated oxygen consumption occurred a t the time when the A-V 0, was markedly diminished. Nitroglycerin invariably diminished oxygen consuinption and blood pressure (Fig. 2).

FIG.4. Effect of angiotensin ( 1 &kg) on coronary circulation and cardiac oxygen consumption. Angiotensin waa given intravenously. Note reduction in A-V 0,but increase in oxygen consumption. Remainder of legend aa in Fig. 2.

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FIQ.5. Effect of histamine (2 pg/kg) on coronary circulation and cardiac oxygen consumption. Histamine was given intravenously. Note biphasic change in oxygen consumption. Over-all change was an increase. Remainder of legend aa in Fig. 2.

Coronary flow usually decreased or remained unchanged and A-V O2 changed only slightly. Dipyridamole increased oxygen consumption slightly, increased coronary blood flow markedly, and narrowed A-V 02. 3. Conclusion Myocardial oxygen consumption furnishes an important parameter required in the evaluation of any antianginal agent; the A-V O2alone is not adequate. Continuous recording of saturation of arterial and sinus blood, which is possible in animals, has certain advantages in that rapid changes in oxygen consumption can be observed. These may be limiting to the use of the compound. If coronary dilatation cannot occur in the anginal patient any increase in oxygen consumption, even if for a short period, would be undesirable. The method might be improved by perfusing blood into the coronary artery a t a constant rate, thereby tending to resemble the anginal situation.

C. MEASUREMENT OF MYOCARDIAL OXYGENTENSION 1. Method In view of the fact that coronary insufllciency is believed to result from an imbalance between the oxygen supply and demand a t the tissue level, the determination of tissue oxygen tension (02 tension) may be an important approach to the evaluation of antianginal drugs. The 0, tension of the tissues (muscle cells) is dependent upon the rate of arterial blood flow (actually capillary flow), the O2tension of the plasma and the

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rate of diffusion of 0, from the plasma to the muscle cells. Diffusion rate is related to the concentration gradient between the cells and the extracellular fluid. Blood flow really gives an indication of the rate of oxygen availability per unit time (blood flow in ml/min X 0, content of arterial blood in ~ 0 1 % ) . The use of the polarographic method for the study of myocardial oxygen tension started with the work of Sayen et al. (1951). Since that time numerous reports have been published by that group, but we will only consider those related to the present discussion of the technique-the advantages and limitations. Oxygen tension is measured with an open-tip platinum electrode which is inserted to a depth of about 2 mm into the myocardium of an openchest dog. Since the electrodes are small, 0.4 to 0.6 mm in diameter, several can be placed in the heart a t one time. A limb is usually placed in a saline bath which is connected to a calomel electrode by a salt bridge thereby completing the circuit. Electrolysis is carried out a t 0.6 v and the relative oxygen tension is read from a sensitive galvanometer or recorded continuously. This is the basic procedure used by most investigators. Occlusion of a branch of a coronary artery results in an immediate reduction in the 0, tension of the dependent area (Sayen et al., 1951,1958, 1960; Miyashita, 1962). The electrocardiographic changes (ST segment elevation) in the ischemic area appear considerably later than the reduction in 0, tension (Sayen et al., 1958; Miyashita, 1962). When the animal breathes pure oxygen there is a marked increase in the 0, tension of the normal area but little change in the central zone of the ischemic area, and the electrocardiographic change is not altered (Sayen et al., 1951, 1958, 1960; Miyashita, 1962). Similarly, norepinephrine intravenously produces a rise in the 0, tension of the normal tissue with no change in the ischemic zone (Sayen et ul., 1952, 1960; Miyashita, 1962). Breathing 10% oxygen lowers 0, tension in the nonoccluded areas (Sayen e t aZ., 1960). After partial occlusion there is a decline in 0, tension but the area still shows a response to 10 and 100% oxygen inhalation or norepinephrine (Sayen et al., 1960). A recent study with norepinephrine is of particular interest in that it showed a distinction between the changes in oxygen tension and the electrocardiogram (Sayen e t uZ., 1960). I n the normal areas there was ST segment displacement, increased contractility, and a concomitant increase in O2tension. I n severely ischemic areas (central zone of occluded area), norepinephrine produced some alteration of the electrocardiogram (partial reversal of ischemic pattern), but 0,tension remained a t low levels and contractions were not altered. The authors concluded that the ST segment deviations due to norepinephrine do not indicate ischemia but are closely associated with the positive inotropic effects and an increased

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oxygen supply/demand ratio (Sayen et aZ., 1960). These results can be interpreted differently because of the limitations of the technique. The electrodes do not really reflect what is occurring a t a cellular level but rather indicate oxygen delivery to the tissues by the blood. Norepinephrine increases myocardial oxygen consumption, and it may well be that cellular hypoxia develops because of some metabolic change in spite of the plethora of oxygen. The electrocardiographic changes may be associated with cellular hypoxia and loss of electrolytes. We have carried out studies with the polarographic technique over several years in an attempt to determine if this would be a more rational approach to evaluation of antianginal agents. Theoretically, the electrodes show changes in the supply/demand ratio to the tissues, and this seems to be the case. Unfortunately, when the blood flow is permitted to change, the electrodes reflect primarily the changes in the supply and give no indication of changes in myocardial metabolism. We were able to duplicate the previously discussed effects on 0, tension of occlusion of an artery, of breathing 10 or 100% oxygen, or of injection of norepinephrine in both the dog and miniature pig. Nitroglycerin given intravenously reduced the 0, tension of the left ventricle but when given directly into the coronary artery, 0, tension rose. Direct measurement of coronary blood flow rate demonstrated a close correlation between blood flow and tissue 0, tension when flow was altered manually (screw clamp) or by drugs, indicating that when blood flow can vary, the 0, tension is primarily a reflection of tissue availability. When the animal was allowed to breathe 100% oxygen instead of room air, there was a latent period of 10 to 20 seconds before the 0, tension increased. It was felt that nitroglycerin might shorten this latent period; however, intravenous administration did not alter the delay in normal areas or areas made slightly ischemic by partial occlusion of a coronary artery. Another approach was based on the premise that nitroglycerin may promote increased collateral blood flow and thereby improve the 0, supply to a compromised area. To perform these studies, the 0,tension of a small area was reduced to about 50% of the control value by partial occlusion of the artery, and nitroglycerin was administered intravenously. The 0, tension fell in the ischemic area as well as in the normal area. Another group (Honig et aZ., 1960), using a permanently implanted oxygen electrode, studied the effect of nitroglycerin in intact dogs and found an increased 0, tension which was interpreted to indicate an increased blood flow. It would appear this technique could be a valuable tool if 0, availability was maintained a t a constant level. I n this way, the tissue 0,

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tension would be dependent upon changes in demand. This could be approached by perfusing the coronary arteries a t a constant rate, which would keep 0, availability constant. Then the changes in O2tension would reflect changes in tissue utilization. Some preliminary attempts were carried out, but because of the use of anticoagulants there was bleeding a t the site of electrode insertion so that the values for tissue 0, tension were incorrect. However, if this aspect of the problem could be solved, such an approach might be a valuable tool for evaluation of antianginal agents. 2. Conclusion

The polarographic technique for measuring tissue 0, tension by means of oxygen electrodes really measures availability of oxygen which is dependent upon blood flow and plasma 0, content. It does not reflect what is occurring within the cell. Intracellular recordings might be very valuable but such techniques have yet to be developed. If the rate of coronary blood flow were constant, the O2 tension of the tissues would be altered only by changes in oxygen requirements. This might be an approach to the study of oxygen consumption a t a tissue level.

D. EXPERIMENTAL CORONARY INSUFFICIENCY INDUCED BY CORONARY OCCLUSION OR DRUGS It may well be that the failure in transposing the laboratory pharmacology on the coronary circulation to the clinic is due to the fact that the pharmacological studies have been carried out in animals with a normal coronary circulation, which differs considerably from the coronary circulation in the patient with coronary insufficiency. Experimental coronary insufficiency can be induced in animals in a number of ways but there is always the question as to how closely the experimental procedure resembles the clinical situation. Several of the procedures have used the electrocardiogram as the basis of evaluation; the changes induced frequently resemble those that occur during an acute anginal episode in man. The similarity of the electrocardiographic changes does not indicate that the same fundamental mechanism obtains, but a t least the models have an abnormality of the coronary circulation somewhat akin to that in man. The approaches that will be considered are coronary occlusion, vasopressin-induced spasm, ergotoxine-induced ST segment alteration, and insufficiency induced by atherosclerosis. 1. Coronary Occlusion

Abrupt total occlusion of a branch of the left or right coronary artery of the dog, cat, or pig results in a marked ST segment elevation in the

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surface electrocardiogram from the ischemic area and an ST segment depression in limb leads (DiPalma, 1961; Raab et al., 1962; Varma and Melville, 1962a; Winbury et al., 1962b). Similar changes have been observed even after partial occlusion (reduction in diameter of 50 to 75% of normal) of the descending branch in the pig (Winbury et al., 1962b). I n the dog, a reduction of blood flow of 35 to 70% in the descending branch is required to produce slight ST segment changes; with a greater reduction in flow the ST segment elevation (surface lead) is marked (WQgria et al., 1949). a. Epicardial electrocardiographic recordings. DiPalma (1961) used the rate of development of the ischemic electrocardiogram after occlusion of a coronary artery for the evaluation of the coronary circulation. The rate at which the ischemic changes appear is assumed to depend mainly on the collateral circulation available to the compromised area and the state of cardiac metabolism. A marginal branch of the right coronary artery was occluded and the time required for the maximal degree of elevation of the ST segment in the ischemic area was determined (ischemia time) ; recovery time after release of occlusion was also determined. It was observed that the ischemia time varied inversely with blood pressure and heart rate, whereas recovery time varied directly with these parameters. Thus when the work of the heart was increased (increased heart rate or blood pressure), ischemia developed more rapidly and recovery took longer. For example, doses of norepinephrine which increased heart rate and blood pressure shortened ischemia time and lengthened recovery time. Hexamethonium prolonged ischemia time and did not alter recovery time. It was concluded that hexamethonium reduced the work of the heart relatively more in proportion to the reduction in coronary flow. I n order to take into account the effect of a drug on the work of the heart and be able to evaluate the supply/demand ratio, the author used the ischemia index which is ischemia time X blood pressure X heart rate. A change in the index should indicate a change in the balance between coronary blood flow and cardiac muscle metabolism. Sympathomimetic amines (norepinephrine, metaraminol, and methoxamine) did not significantly affect the ischemia index or the recovery index; likewise, angiotensin I1 and sodium nitrite did not affect the indexes. This method might provide a new approach for the evaluation of antianginal agents but I wonder if the ischemia time or ischemia index is more significant, because we may well be seeking a compound which reduces the work of the heart. This would prolong ischemia time but may not change the ischemia index. It would be of interest to study ischemia time and index a t a fixed coronary blood flow rate, which is similar to the situation in the anginal patient.

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We have used a somewhat different approach in the dog and determined the degree of reduction in coronary blood flow required to induce epicardial ST segment elevation before and after a drug. There is considerable variability within an animal over a period of time so that it is difficult to determine if a compound produces a significant effect. Perhaps if the changes in blood pressure and heart rate were taken into account, and an index similar to that proposed by DiPalma (1961) calculated, the variability would be reduced; the ischemia index has been found to be much less variable than the ischemia time. Varma and Melville (1962a) noted that the ST segment alteration was variable during the first hour after total occlusion of the anterior descending branch in the dog, but the pattern remained more consistent for the following 2 to 3 hours. Hypoxemia (breathing 10% oxygen) produced a further depression of the ST segment (lead 11). Intravenous administration of nitroglycerin, trolnitrate, aminophylline, papaverine, dipyridamole, or N - [ 3'-phenyl propyl- ('2') ] -1,l-diphenyl propyl- (3)amine (Segontin) after 1 hour of coronary occlusion enhanced the ST segment depression whether the animal was breathing room air or 10% oxygen. Thus the ST segment deviation produced by coronary occlusion was not favorably modified by coronary dilator drugs including nitroglycerin, indicating that this procedure would not be suitable for evaluation of antianginal agents. Using vagotomized cats, Raab et al. (1962) occluded the anterior descending branch to a point just short of producing ST segment displacement of the surface electrocardiogram. General hypoxia (nitrogen breathing) for 2 minutes produced maximal ST segment elevation in the animals with a restricted coronary flow, but no change in animals with normal coronary flow. Similar effects were produced by stimulation of the cardiac sympathetic nerves, intravenous injection of norepinephrine, or epinephrine, or by stimulation of the muscles of the hindlimb only when there was coronary restriction. There were no studies on nitrites or other antianginal agents since the primary objective was to demonstrate the importance of the catecholamines in the development of the electrocardiographic changes. The procedures described would be worthwhile to investigate with antianginal agents and adrenergic-blocking agents, but until this is done we must withhold judgment on the relative merits of this approach, particularly in view of the negative results with nitroglycerin following coronary occlusion and hypoxemia in the dog, which were previously discussed (Varma and Melville, 1962a). b. Chronic occlusion. Acute partial or total occlusion of a major branch of the left coronary artery of the anesthetized pig frequently results in ventricular fibrillation within 1 hour (Winbury et al., 1962b).

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Electrical defibrillation procedures were successful in a high percentage of the cases and the animals recovered after closing the chest and proper postsurgical care. I n our experiments, anesthetized dogs are not as vulnerable to ventricular fibrillation following abrupt total occlusion, and partial occlusion of the anterior descending branch has no effect. These results are at variance with those of Harris (1950), who reported a high incidence of fibrillation following abrupt total occlusion of the descending branch; partial occlusion 30 minutes prior to the total occlusion prevented the fibrillation. Regelson and co-workers (1959) studied the effect of iproniazid on the survival of conscious dogs following acute coronary occlusion. One week prior to the experiment a loose ligature was placed around the anterior descending coronary artery. Occlusion was produced by tightening the ends of the ligature that had been passed through the chest wall. I n the control group, 67% died within 1 hour after occlusion due to ventricular fibrillation, but in the group treated with iproniazid daily for 4 days prior to the occlusion only 21% succumbed to ventricular fibrillation. This effect does not appear to be specific since pentobarbital anesthesia, morphine, reserpine, hexamethonium, and chlorpromazine also reduced the incidence of fibrillation. In all probability the protective effects involve some form of autonomic blockade or depression of myocardial excitability, and it is rather unlikely that this type of procedure would be suitable for evaluation of antianginal agents. We have attempted, without much success, to induce electrocardiographic changes by stress in unanesthetized pigs and dogs with a compromised coronary circulation. Pigs subjected to hypoxemia (10% oxygen) for 30 minutes a t various times during the first 2 weeks following acute, partial or total occlusion of the anterior descending coronary artery did not show any specific alteration in the electrocardiogram. Likewise, exercise of pigs or dogs with partial or fotal occlusion did not produce diagnostic electrocardiographic changes. Further, the exercise tolerance was not significantly reduced. Asada et al. (1962) carried out some quantitative studies on the effect of hypoxemia in dogs with progressively developing coronary occulsion. A gelatin sponge saturated with dicetyl phosphate was placed around the anterior descending branch; gradual narrowing of the lumen occurred over 4 to 6 weeks and was maintained for several months to 1 year. Hypoxemia tests under pentothal anesthesia were used to evaluate the degree of coronary insdciency. The oxygen content of the inspired air was gradually lowered until some positive electrocardiographic findings appeared. At this point the oxygen saturation of the arterial blood was determined. Any of the following was considered a positive response: (1) ST segment dis-

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placement of more than 0.1 mv; (2) T flattening of more than 0.15 mv, or (3) abnormal rhythm. The oxygen saturation a t the point of a positive electrocardiographic response was 71% for dogs with coronary occlusion compared with 49% for normal dogs. Animals treated with dipyridamole for 30 to 280 days, starting on the day after coronary occlusion, had an arterial saturation of 58% a t the point of a positive response. The authors did not perform any experiments on animals to whom drug was given only after the arterial narrowing was established, This approach to the production of chronic coronary insufficiency appears to offer potential, using the degree of desaturation of the arterial blood required for positive electrocardiographic changes as the end point. Determination of the effect of drugs on an acute or chronic basis after the arterial lumen has been reduced would have similarity to the clinical situation. Theoretically, an increase in the tolerance to hypoxemia could be effected either by a hemodynamic or metabolic change or a combination of both. 2. Electrocardiographic Changes Produced by Pituitrin Posterior pituitary extract (pituitrin) and vasopressin produce coronary constriction in the isolated heart and intact animal (WBgria, 1951; Winbury and Green, 1952; Hashimoto et al., 1960; Karp et al., 1960; Winbury et al., 1962a). Studies on Rbs6uptake by the myocardium suggested a decrease in “effective capillary blood flow” (Love and Burch, 1957a,b ; Winbury et al., 1962a) ,but there appeared to be no change in the oxygen consumption of the isolated fibrillating heart (Hashimoto et al., 1960). Thus pituitrin leads to coronary insufficiency and electrocardiographic alterations because of inadequate blood flow. Lindner and co-workers (1953) described a method for evaluation of agents in the intact dog, giving pituitrin intravenously before and after the drug. The significant changes in the electrocardiogram were an elevation in the amplitude pf the T wave and extrasystoles. Recosen, an extract of fresh heart, which has coronary dilator properties, given before the pituitrin prevented the electrocardiographic changes, A similar procedure was used in anesthetized rabbits for the evaluation of dipyridamole (Mutti and Chiari, 1961) and in rats for the evaluation of monoamine oxidase inhibitors (Cahn and Herold, 1962). Both dipyridamole and isocarboxazid attenuated or prevented the electrocardiographic alterations produced by pituitrin (or vasopressin). This approach seems rather nonspecific, since any agent which is an effective coronary dilator should prevent the coronary constriction induced by pituitrin. Moreover, it is rather unlikely that spasm of the coronary artery is involved in the majority of patients with coronary insufficiency (Section I).

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3. Electrocardiographic Alterations Produced by P k o t o d n Injection into the Lateral Ventricle Varma and Melville (1962a) showed that injection of 0.1 mg of picrotoxin into the lateral cerebral ventricle of the rabbit produced ST segment depression and cardiac irregularities. The animals were anesthetized, vagotomized, curarized, and under artifical respiration. The effecb of picrotoxin usually persisted for 30 to 60 minutes. Nitroglycerin, trolnitrate, and dipyridamole reversed or reduced the ST segment depression, whereas aminophylline and Segontin enhanced the ST segment depression. Iproniazid had no effect on the ST segment depression. This method seems to have some promise on an empirical basis, but it is important to learn more about the hemodynamic changes produced by the injection of picrotoxin into the lateral ventricle before any conclusion can be reached.

E. EXPERIMENTAL CORONARY INSUFFICIENCY INDUCED BY ATHEROSCLEROSIS Atherosclerosis has been studied extensively from the standpoint of endocrinology, nutrition, biochemistry, and pathology. Few physiological studies have been carried out on atherosclerotic animals, and i t would appear a useful means of attempting to duplicate an experimental model of coronary insufficiency. This, of course, would require evidence, other than pathological, that the coronary circulation is compromised. 1. Stress Tests in Atherosclerotic Rabbits

Rinzler et al. (1955, 1956) and Karp et al. (1960) demonstrated that ergonovine maleate produced an ST segment depression in Dutch belted male rabbits on a 2% cholesterol diet. All rabbits with a positive ST segment depression had extensive coronary atherosclerosis. Studies by Stein (1949, 1963) on a large number of anginal patients clearly demonstrated that ergonovine maleate produced significant ST segment depression and pain similar to that observed during an anginal attack or induced during the exercise (Master et al., 1944) or anoxia (Levy et al., 1941) test. The pain and electrocardiographic abnormalities were relieved by sublingual administration of nitroglycerin, indicating that coronary insufficiency was involved. It has been assumed that ergonovine induced constriction of the coronary arteries in these anginal patients. At the time of the original report on the use of ergonovine (Stein, 1949) for diagnosis of angina, we studied the effect of ergonovine on normal dogs, but could find no evidence of coronary constriction (coronary flow) nor any electrocardiographic changes

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(ST segment depression) following intravenous administration of ergonovine to unanesthetized dogs with chronic partial occlusion of the anterior descending branch of the left coronary artery. It was not until the report of Karp and co-workers (1960) that there was experimental evidence suggesting that ergonovine produces coronary constriction. These studies are of particular importance because they demonstrate for the first time a difference in the pharmacological response of the normal heart and the atherosclerotic heart. I n isolated atherosclerotic rabbit hearts perfused by a modified Langendorff procedure, ergonovine frequently produced a decrease in coronary flow (coronary constriction) and did not produce a significant increase in coronary flow. On the other hand, similar doses produced virtually no change or coronary dilatation in normal hearts. Quantitative differences were noted in the response of the normal and atherosclerotic heart to other vasoactive agents. This will be discussed below. The ergonovine test in atherosclerotic rabbits appeared to be an excellent approach for the evaluation of agents for use in coronary insufficiency since the model closely duplicates the situation in the human with angina. Accordingly, investigations were initiated on the use of this technique by Winbury et al. (1961), but i t was found that less than 30% of the atherosclerotic rabbits (Dutch belted) showed a significant electrocardiographic response to ergonovine even after 6 months on the 2% cholesterol diet. Use of Levy e t al. (1941) anoxia test instead of the ergonovine test markedly increased the sensitivity of the procedure so that after approximately 4 months on the 2% cholesterol diet 74% of the rabbits showed a consistent ST segment depression when subjected to anoxia. To perform the hypoxia test the rabbits were lightly narcotized with pentobarbital sodium and a small chamber placed over the head. A mixture of 10% oxygen in nitrogen was passed through the chamber for 10 minutes and the animal then permitted to breathe room air. Electrocardiograms were recorded at, intervals before, during, and after the hypoxia test using lead I1 and a mid-sternal chest lead ( V) . ST segment depression of 0.75 mm (1 mv = 10 mm) was considered a positive response. Normal animals subjected to hypoxemia for 10 to 30 minutes did not show such changes. I n atherosclerotic animals S T segment depression occurred within 1 to 2 minutes after anoxemia was started and disappeared within 1 minute after return to room air. After a positive response to hypoxemia was established continued feeding of the 2% cholesterol diet was not essential. Animals that were returned to the control diet (no added cholesterol) continued to display a positive response to hypoxia for a t least 3 months. However, after this period responses were variable and by 5 months after withdrawal of cholesterol a positive response to hypoxemia was absent in spite of the same coronary pathology as animals continu-

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ously on the diet. All of the animals on the 2% cholesterol diet showed similar pathological changes consisting of marked atherosclerotic involvement of the coronary arteries, aorta, pulmonary arteries, and of the aortic valves and mitral valves. In addition, there was a varying degree of myocardial damage. It is important to note that there was no obvious relationship between the electrocardiographic response to hypoxemia and the pathological changes. Varma and Melville (1962b) were able to duplicate the procedure previously described and affirmed that a positive or negative response in atherosclerotic animals does not appear to be related to the degree of pathology. The sensitivity of the procedure was improved by Tabachnick and co-workers (1961) by using a gas mixture containing 6.5% 02: 4.5% CO, :89% N, (Coulshed, 1960). Another modification proposed was the use of ergonovine maleate 0.05 mg/kg intravenously while the rabbits are breathing 10% oxygen (Varma and Melville, 1962b). With this combined procedure 100% of atherosclerotic rabbits showed a positive response. Reserpine pretreatment intensified the ST depression following hypoxemia (Melville and Varma, 1962). There is no doubt that the hypoxemia procedures (Tabachnick et al., 1961; Winbury et al., 1961; Varma and Melville, 1962b) have many advantages over the ergonovine procedure (Rinzler e t al., 1955, 1956; Karp et al., 1960) because of increased sensitivity, ease of performance, and rapidity of response. Various drugs used in the treatment of coronary insufficiency were evaluated in atherosclerotic rabbits using the hypoxemia test. Nitroglycerin (5 to 40 pg/kg, intravenously), or aminophylline (10 mg/kg, intravenously) were ineffective in relieving or preventing the ST segment depression induced by hypoxemia (Winbury et al., 1961). Other agents found ineffective were trolnitrate (1 to 2 mg/kg, intravenously), papaverine (1 to 2 mg/kg, intravenously), dipyridamole (0.5 mg/kg, intravenously), and Segontin (0.25 to 1 mg/kg, intravenously) (Varma and Melville, 1962a). Thus it can be seen that agents which produce coronary dilatation do not prevent the response of atherosclerotic rabbits to hypoxemia. Likewise, monoamine oxidase inhibitors such as iproniazid or pheniprazine did not prevent the hypoxemia response (Tabachnick et al., 1961; Varma and Melville, 1962a). Concurrent feeding of propylthiouracil with the cholesterol reduced the severity of the hypoxemic ST segment depression (Tabachnick et al., 1961). Of great significance are the results of Tabachnick et al. (1961) on triparanol. Treatment of rabbits with a well-established hypoxemia response with 25 or 50 mg/kg, intraperitoneally, daily for 2 weeks reduced the ST segment depression while simultaneous vehicle controls showed no improvement. When therapy was discontinued, the hypoxemic ST segment depression gradually reverted to the pretreatment pattern. These findings correlate well with the clinical obser-

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vation that this agent prevented the ST segment alteration produced by exercise in the anginal patient (Hollander et aZ., 1960). It can be concluded that although the response of atherosclerotic rabbits to hypoxemia resembles that of the anginal patient, there must be a fundamental difference, since a nitrate such as nitroglycerin which is highly effective in man did not prevent the hypoxemic ST segment depression in atherosclerotic rabbits (Winbury et al., 1961; Varma and Melville, 1962a). Winbury et al. (1961) raised the question about the mechanism of the coronary insufficiency in these rabbits on the basis of the gross and microscopic pathology observed. The fact that there was considerable myocardial damage and valvular incompetence and insufficiency in addition to the coronary atherosclerosis would suggest an additional factor of myocardial failure. When the stress of hypoxemia increases the circulatory load the heart may not be capable of responding sufficiently to maintain homeostasis. Nevertheless, the results with triparonol indicate that some agents can prevent the electrocardiographic changes (Tabachnick et al., 1961). This method may offer an approach to the “in vivo total metabolic” evaluation of agents for the treatment of coronary insufficiency and enable the development of antianginal agents with an entirely unique mechanism of action, primarily based on a change in myocardial metabolism. 2. Response of Atherosclerotic Heart to Vmoactive Agents Pharmacological studies on atherosclerotic hearts have been limited. Certainly the results of Gorlin et al. (1959a) demonstrate that there is a difference in the response of the normal and anginal patient to nitroglycerin. This suggests that studies on atherosclerotic hearts may indicate different pharmacology. Karp et al. (1960) compared isolated hearts from normal and atherosclerotic rabbits using a modified Langendorf technique. The atherosclerotic heart had a higher basal coronary flow rate and a smaller contraction amplitude than normal hearts. Melville and Varma (1962) noted that the atherosclerotic heart was heavier than the normal heart (9.7 versus 6.3 gm) ; when the coronary flow rate was calculated on a unit weight basis (CBF/gm), the normal group was higher than the atherosclerotic group. Qualitative differences in the response of the normal and atherosclerotic heart (constriction) to ergonovine were previously discussed (Section V, E, 1). Nitroglycerin produced a greater percentage increase in coronary flow in the normal than in the atherosclerotic heart, and the maximum flow achieved was greater (Karp et al., 1960). The greater response of the normal heart was also noted after papaverine and aminophylline (Melville and Varma, 1962). However, there was no dif-

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ference in the response to trolnitrate (Melville and Varma, 1962). Likewise vasopressin (0.008-0.8 U) produced a profound decrease in coronary flow in normal and atherosclerotic rabbit hearts which could be reversed by nitroglycerin (Karp e t al., 1960). Cross and Oblath (1962) compared the pressure-flow relationship in normal dogs with dogs made atherosclerotic by a diet containing cholesterol, thiouracil, meat, and lard. For the study of coronary function the heart was placed on a bypass pump-oxygenator system and the pressureflow relationship (P/F) studied under control conditions and during infusion of Pitressin ( 5 U/liter) and norepinephrine (15 pg/liter) in blood infused. Under control conditions the P/F curve wm the same in the atherosclerotic and normal dogs. Pitressin produced a greater reduction in the slope of the normal than the atherosclerotic animals ; norepinephrine produced a greater increase in the slope in the normal. These studies lead to the conclusion that under control conditions the P/F relationship is normal in the atherosclerotic dog heart; however the vessels are less responsive to vasodilator or vasoconstrictor agents. The fact that the atherosclerotic heart may well respond differently than the normal heart has been demonstrated in rabbit, dog, and man, and emphasizes the importance of understanding the physiological basis of the disease. Further studies on atherosclerotic hearts are needed in order to better define the physiological and pharmacological differences, and i t may well be that this is a better preparation for evaluation of antianginal agents than the normal heart.

3. Conclusion-Experimental

Coronary Insufficiency

There are a variety of approaches to the production of coronary insufficiency. These include coronary occlusion, pituitrin vasospasm, and atherosclerosis. The coronary insufficiency produced by means of these procedures is based on alterations in the electrocardiogram which resemble those used for diagnostic purposes in man (ST segment or T wave changes). The model which appears t o show a close similarity to the clinical disease is the atherosclerotic rabbit. Hypoxemia stress tests in such animals produce ST segment depression ; however, prophylactic or therapeutic treatment with nitrites or other coronary vasodilators do not prevent these hypoxemia-induced changes. Nevertheless, this model could be of great value since it permits evaluation of the effect of agents of myocardial 0, metabolism. Hypoxemia reduces O2 availability to the myocardium, producing insufficiency, and an agent which would prevent the electrocardiographic changes would do so by preventing or reducing the insuflkiency.

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This beneficial effect could result from a reduction in the work of the heart or by improvement of the biochemical efficiency for energy production. Another model which warrents further investigation is the dog with progressively developing coronary narrowing as a result of local administration of dicetyl phosphate. These animals showed an increased sensitivity to the electrocardiographic disturbances produced by hypoxemia. This was quantitated by determination of the degree of arterial desaturation required to produce significant electrocardiographic changes. An agent which would permit a greater degree of hypoxemia before such electrocardiographic changes occur might have potential in the treatment of angina. Such an effect could result from a reduction in the cardiac 0, requirement, increased collateral coronary flow, or a cardiac metabolic change. These two models also have the advantage of permitting the chronic use of the animals. Other procedures which seem of interest and should be studied further are (1) determination of time to produce ischemic electrocardiogram after acute coronary occlusion in the dog; (2) anoxia-induced electrocardiographic changes in the cat with partial occlusion of a coronary artery; and (3) electrocardiographic abnormalities produced by injection of ergotoxine into the lateral cerebral ventricle.

F. PROLONGATION OF CONTRACTILE ACTIVITY DURING ANOXIA Anoxia of the isolated atria, papillary muscle, or isolated heart results in a rapid reduction in contractile activity (Winbury, 1956; Setnikar and Ravasi, 1960; Siess, 1962). Reintroduction of 0, permits full recovery of contractile amplitude, providing the period of anoxia was not prolonged and adequate substrate was present (Winbury, 1956; Setnikar and Ravasi, 1960). The biochemical consequences of anoxia have been discussed in detail in Section 11, E and will not be considered here. In any event, an agent which permits a longer duration of contractile activity under anoxia might be considered to have an influence on myocardial metabolism. Isolated rabbit hearts were used to determine the effect of iproniazid on contractile activity under anoxic conditions (Setnikar and Ravasi, 1960). With iproniazid present in the perfusion medium the period of anoxic contractions was increased. Likewise, hearts from animals treated for 7 days wit.h iproniazid were capable of more prolonged activity during anoxia compared with untreated controls. During anoxia of the electrically driven isolated atria from the guinea pig or rat, contractile activity ceases and there is little response to epinephrine or strophanthin (Siess, 1962). Pretreatment with dipyridamole does not prevent the arrest of activity during anoxia but does permit the

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full response to epinephrine and strophanthin. Dipyridamole prevents the normal decline of ATP due to anoxia and it has been assumed that this permits the response to epinephrine or strophanthin. The question arises as to whether or not these methods reflect properties of compounds that might be involved in antianginal action. Certainly we know that iproniazid may relieve the pain but does not improve the basic coronary insufficiency. Furthermore, there is question about the clinical effectiveness of dipyridamole. Until more data have been accumulated on the actions of nitrites and other drugs, no conclusions can be drawn.

0. ANTAGONISM OF EFFECT OF CATECHOLAMINES ON THE HEART The catecholamines increase myocardial oxygen consumption in excess of that required for the increased cardiac work (Section 11, C ) . Thus there is a decline in cardiac efficiency. Not only is there an increase in “active” oxygen consumption but “resting” oxygen consumption is increased as well. The increased oxygen consumption occurs if the catecholamine is released from endogenous stores in the myocardium, sympathetic nerve endings (norepinephrine), the adrenal gland (epinephrine), or is injected intravenously or into the coronary artery. Coronary flow increases in response to sympathetic stimulation or injection of catecholamines. The increase in flow frequently more than compensates for the increased oxygen consumption and oxygen availability to the myocardium rises. This results in an increase in myocardial 0, tension, an increase in coronary sinus 0, saturation, and a decrease in A-V 0,. Under these circumstances the O2supply/demand ratio actually improves and may not indicate what is taking place at the cellular level. When the coronary blood flow cannot rise, the coronary sinus 0, saturation declines and A-V 0, increases in response to catecholamines. The direct action of norepinephrine and epinephrine on the coronary vessels is vasoconstriction which becomes evident when the metabolic actions are blocked. In addition to the anoxiating action of norepinephrine there is a reduction in effective capillary blood flow. Both of these actions would lead to coronary insufficiency when the coronary circulation is compromised and vasodilatation is limited. Raab and co-workers (1950, 1956, 1962) emphasized the role of the catecholamines in angina and postulated that nitroglycerin may antagonize the catecholamine-induced increase in myocardial oxygen consumption. This could not be confirmed by others (Eckstein et al., 1951; Popovich e t d., 1956; Winbury et al., 1962a). Nevertheless, recent clinical studies have demonstrated that a p-adrenergic-blocking agent, nethalide, has a beneficial effect in anginal patients (Dornhorst and Robinson, 1962). This is

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a most important development and lends support to the role of the catecholamines in angina. It seems likely to suppose that p-adrenergic-blocking agents may provide a new and more logical approach to the treatment of coronary insufficiency than coronary dilators. This is particularly significant in view of the suggestion that the coronary bed of the anginal patient is incapable of vasodilatation and the observed rise in blood catecholamine levels in anginal patients during exercise. 1. Adrenergic Blockade

The inotropic and chronotropic actions induced by the catecholamines or by cardiac sympathetic stimulation can be antagonized by p-adrenergic-blocking agents (dichloroisoproterenol or nethalide) but not by aadrenergic-blocking agents (phenoxybenzamine, phentolamine, piperoxan, azapetine, Hydergine, or dibenamine) (Moran and Perkins, 1958, 1961 ; wickerson and Chan, 1961; Black and Stephenson, 1962). The increase in oxygen consumption produced by epinephrine, norepinephrine, and isoproterenol is antagonized by dichloroisoproterenol (Hashimoto et al., 1960). Although there are a number of ways of evaluating p-adrenergicblocking activity, i t would be most logical to use the heart for the initial screen. It is conceivable that a compound may have some greater specificity of action for the heart than another organ system, which would be most desirable (Black and Stephenson, 1962). Blockade of the inotropic or chronotropic action of the catecholamines in vitro or in vivo is a desirable parameter but more to the point is the blockade of the increased oxygen consumption (Hashimoto et al., 1960). I n addition, it would be important to determine if agents will antagonize the reduction in effective capillary flow induced by norepinephrine. 2. Myocardial Necrosis Induced by Isoproterenol or Stress

Rona et al. (1959) and Chappel et al. (1959) demonstrated that administration of large doses of isoproterenol to rats on each of two consecutive days resulted in extensive necrosis of the left ventricle. No doubt this effect is due to the intense and prolonged cardiac stimulation. This procedure was used for the investigation of a large group of compounds of diverse pharmacological action including coronary dilators, monoamine oxidase inhibitors, psychosedatives, and adrenolytics (a) (Zbinden, 1962). The monoamine oxidase inhibitors were the only agents that provided any protection. Raab et al. (1961) presented some evidence that the sympathetic nervous system is involved in the myocardial necrosis produced in rats by stress and fluorocortisol. It was suggested that reflex hypothalamic stimulation leads to general adrenergic discharge and adrenal medullary

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stimulation, causing an increase in plasma catecholamines. The degree of necrosis was reduced by dibenamine, guanethidine, reserpine, or mecamylamine, all of which produce some type of autonomic blockade (Raab et al., 1961).

3. Conclusion Blockade of the actions of the catecholamines on the heart appears to be one of the most promising new approaches to antianginal therapy. New agents which can antagonize p-adrenergic action on the heart (positive inotropic and chronotropic effects, and increased oxygen consumption) should be sought.

H. ARTERIOGRAPHIC TECHNIQUES 1. Method

Coronary arteriography permits visualization of the coronary vascular system in the intact animal. I n addition to providing a means for visualization of narrowing or obstruction of a coronary artery, which is useful in confirming clinical diagnosis, the effect of drugs on the caliber of the coronary arteries can be observed in both man and animals. There are many technical modifications but the basic procedure is the same in all cases, namely, injection of an X-ray contrast medium into the root of the aorta or into selected coronary arteries and taking frequent or serial X-ray photographs after the injection. I n some cases the contrast medium has been injected into the aorta during acetylcholine arrest or temporary occlusion of the aorta by an inflatable balloon and then flushed through the coronaries during the first few contractions; in others the injection into the aorta was given automatically during a specific phase of the cardiac cycle. The most useful injection technique is that of direct injection into specific branches, using the catheterization technique described by West and Guzman (1959). Direct injection of coronary arteries has also been used in man for evaluation of drugs (Likoff et al., 1962). All of the coronary dilator agents studied including nitroglycerin, amyl nitrite, isosorbide dinitrate, erythrityl tetranitrate, dipyridamole, aminophylline, papaverine, and Segontin produced an increase in the caliber of the coronaries of dog or man (West and Guzman, 1959; Gensini et al., 1962; Haight et al., 1962; Likoff et al., 1962; Soloff et al., 1962). The effect of other pharmacologically active agents on the caliber of the coronary arteries, as determined by direct visualization following intracoronary injection, is similar to that demonstrated by direct measurement of the changes in coronary flow rate. For example, isoproterenol, epinephrine,

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MARTIN M. WINBURY

norepinephrine, acetylcholine, methacholine, hypoxia, and sodium cyanide increase the caliber of the coronary arteries while Pitressin causes a decrease in the caliber (West and Guzman, 1959). This suggests that coronary arteriographic techniques show the same properties of drugs as demonstrated by coronary blood flow measurements. This is certainly true for normal animals and man; however, there appears to be a dichotomy in the anginal patient, since nitroglycerin increases the caliber of the atherosclerotic vessel (Likoff et al., 1962) but does not increase the rate of coronary blood flow measured by the nitrous oxide technique (Gorlin et al., 1959a). One group of investigators (Likoff et al., 1962) emphasizes that an increase in the caliber of the coronary arteries does not imply an improvement in blood flow. 2. conclusion

Coronary arteriographic techniques permit visualization of the larger vessels under relatively normal conditions. However, it is questionable as to whether or not the results obtained by these techniques have any greater significance than those obtained by measurement of blood flow. The smaller vessels that are involved in the regulation of blood flow cannot be well visualized.

I. USE OF RADIOACTIVE TRACERS With the development of equipment of detection of radioactivity in the blood stream and tissues, a number of new techniques using radioactive tracers have been proposed for the measurement of regional blood flow. The tracers used have included nondiffusible substances such as P'albumin and diffusible materials such as Rbs6, RbS4,K'?, NaZ4,and P i * . External counting techniques for determination of coronary blood flow in humans and animals are being developed but there are still many problems, both theoretical and practical, in their use. Some of t.he procedures depend upon retention of the isotope in the blood, i.e., 1131-albuminor red cells labeled with C P , and estimation of dilution curves. Others depend upon the appearance or disappearance of a diffusible isotope in the heart using the activity time curve as the estimate of flow rate. Since the uptake or removal of a rapidly diffusible substance by an organ is dependent has pcrupon plasma flow through capillary beds, the use of Rbs6 and K42 mitted study of blood flow which serves an actual nutritional purpose in transport of solute from plasma to the tissues. It is such use of isotopes that may provide a new approach to the evaluation of antianginal agents since the nutrition of a tissue is dependent upon effective capillary blood flow (nutritional flow),

DEVELOPMENT OF ANTIANGINAL DRUGS

67

1. NondiffusibleTracers

The indicator-dilution principle has been applied to the estimation of cardiac output for many years and the same concepts have been applied to the measurement of blood flow through various organs. If a nondiffusible indicator is injected into an organ system, a dilution curve will be obtained from the venous blood, the area under which will be dependent upon the amount of indicator injected and the volume of blood flow. This, of course, assumes adequate mixing and that indicator is not lost or added to in the initial passage through the organ. If the blood flow remains constant, injection of a smaller amount of indicator will yield a curve of proportionately smaller area. This principle would apply if the second curve were generated by direct injection of indicator or by indicator having recirculated through the organ as a result of the first injection (Conn, 1962). Thus, blood flow through an organ can be estimated after a single intravenous injection into man or an animal on the basis of the assumption that the amount of indicator passing through the organ on the initial circuit is related to the fraction of cardiac output passing through that organ. Although this principle seems simple enough, there are many practical problems when applied to measurement of coronary blood flow; these have been considered by Conn (1962). For measurement of the coronary flow it has been assumed that the coronary circuit is the shortest circuit between the aorta and the right ventricle, and that the first curve due to recirculation is related to coronary flow. However, it appears that this may not be the case, since blood containing indicator frequently returns to the right ventricle from the superior vena cava simultaneously with that from the coronary sinus. Thus the derived values for coronary flow exceed the true values. I n spite of the problems encountered with the direct sampling procedures, Sevelius and Johnson (1959) described a procedure using external precordial measurement of activity in right heart blood after injection of 1131-albumin. As with the direct sampling technique previously discussed, the coronary blood flow is related to cardiac output as the portion of injected indicator passing through the coronary circuit. The practical and theoretical problems in the application of this procedure to the human are many and considerably more work is required before there can be general acceptance of the technique (Conn, 1962). 2. Diffusible Tracers

a. Theory. The exchange of materials between the capillary blood and the tissues is essentially 8 flow-limited process (Love and Burch,

68

MARTIN M. WINBURY

1957a, 1959; Renkin, 1959; Conn, 1962). K42and Rbsa have similar kinetic movements and distribution (Love and Burch, 1957a,b; Conn, 1962) and have been used as the tracer substance. I n heart and skeletal muscle, the rate of K exchange between the interstitial fluid (ISF) and intracellular fluid (ICF) is considerably greater than the plasma-ISF exchange rate; therefore, cell membrane transport is eliminated as a rate-limiting factor (Renkin, 1959; Conn, 1962). Further, the amount of K in I C F is so great compared to that in ISF and plasma that an almost infinite sink is provided for the K42 or Rbss which leaves the capillaries. The theoretical aspect of the transport of K42from blood to tissues has been thoroughly analyzed by Renkin (1959), using the gracilis or gastrocnemius muscle of the dog which has been isolated from all nervous and vascular connections and perfused from a reservoir a t various rates with blood from the same animal, A constant arterial concentration of K42 was maintained throughout each flow rate and arterial and venous radioactivity levels were recorded continuously. The venous blood was a t a constant lower level of K42 than the arterial blood indicating constant removal of K4*a t a steady blood flow rate. The extraction ( E ) of the tracer can be computed as follows: E = A - V / A , where A and V represent arterial and venous levels of radioactivity. The product of extraction and blood flow ( Q ) represents the capillary clearance (C) of the tracer: C = QE. Extraction varies inversely with flow rate so that a t very low flows E is almost complete, but as the flow rate increases E declines. Clearance increases with flow rate but not in proportion to it and tends to plateau a t high flow rates. It is beyond the scope of this presentation to discuss the derivation of PS, the permeability-surface area product, which represents the maximal clearance of tracer theoretically attainable a t an infinite flow rate. I n a recent investigation, Laurence et nl. (1963) was able to confirm the inverse relationship between Q and E and the direct relationship between Q and C using a single-slug technique with Rbss. I n this case extraction was computed from the equation E = (total count Rbs6 injected) /(total count Rbs6 recovered). The RbB6 was injected rapidly into the arterial stream and the total RbS8output in the venous blood from the muscle was determined. b. Application to heart. Love and Burch analyzed the dynamics of Rbss uptake by the dog heart (1957a, 1959) and proposed measurement of blood flow on the basis of precordial activity (1957b). An intravenous infusion of RbB8was administered a t a continuously decreasing rate in order to maintain a constant plasma level. Based on the increase in Rbs6

DEVELOPMENT OF ANTIANGINAL DBUGS

69

level of the myocardium, it was concluded that the initial clearance (C) was representative of the rate of plasma flow (Love and Burch, 1957a). Infusion of norepinephrine increased C Rbss and Pitressin decreased C RbsE (Love and Burch, 1957a,b; 1959). Extraction declined during the increase in blood flow induced by norepinephrine; likewise E declined as the specific activity of the myocardium increased (Love and Burch, 1959). A recent abstract from this group (Love and O'Meallie, 1963) presented the following conclusions: (1) extraction varied inversely with the rate of coronary flow and myocardial specific activity; (2) the isotope content of the myocardium after a known period of Rb8* infusion is determined by the total blood flow; and (3) C is influenced by differences in myocardial hemodynamic status, cellular exchange rates, and K concentration. The dependence of C on coronary flow was confirmed by another group using RbS4 (Bennish and Bing, 1962). Others have approached the study of tissue blood flow by measurement of the rate of removal of tracer (NaZ4or P) from the myocardium using precordial counting techniques (Madoff and Hollander, 1961; Salisbury, et al., 1962; Hollander et al., 1963). A small sample of tracer was injected into the myocardium and the half-time (T-S)for the decline in specific activity determined. NaZ4and 1131 were removed from the normal heart a t an exponential rate. T-% was increased by coronary occlusion and decreased by a rise in coronary blood flow. Thus the removal of isotope is related to changes in coronary blood flow or, more specifically, local tissue (capillary) blood flow. The T-1/2 for was determined in normal and anginal humans by percutaneous injection of IlS1 into the left ventricular apex or injection into the myocardium during surgery (Hollander et al., 1963). T-1/2 for normal patients averaged about 1.4 minutes compared with 5.6 minutes for the anginal patients; there was a wide range in the anginal patients (0.8 to 13.5 minutes) so that the difference may not be statistically significant. I n addition, there were regional differences in T-Y2 in the anginal patients, suggesting that there are regional differences in the tissue perfusion rate. This was not the case in the normal individual. Exercise in the anginal patient reduced T-Y2 but nitroglycerin did not change the value. The absence of a decline in T-% following nitroglycerin in the anginal patient suggests that the nitrites may not augment myocardial capillary blood flow. This may have been associated with the decline in blood pressure which could have prevented a change in coronary flow even if coronary dilatation were present. There are limitations with all of these clearance procedures for the determination of the total coronary blood flow (Love and Burch, 1957b, 1959; Conn, 1962; Salisbury et al., 1962) ; however, they do give a meas-

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MARTIN M. WINBURY

ure of effective capillary perfusion which, to my mind, is the more important parameter since this is a n indication of the availability of blood for exchange of materials with the tissues. Because C and E are related to coronary blood flow it would be difficult to analyze the direct action of drugs on effective capillary flow unless blood flow were constant. We elected to utilize the Rbss extraction approach for the study of vasoactive agents (Winbury et d.,1962a) for the following reasons: (1) measurement of total coronary blood flow does not indicate what portion of flow is available for capillary exchange with the tissues and serves a nutritional purpose; (2) an actual increase in nonnutritional flow can divert blood from the tissues and would be undesirable; and (3) the extraction of RbsGby an organ gives some indication of the relationship of effective capillary flow to total flow. A double isotope procedure using RbsE (diffusible) and P1-albumin (nondiffusible) was ut,ilieed in order to avoid the problems involved in collection of total coronary sinus outflow (Fig. 6). Blood was pumped at

To Vena Densitometsr

Collection Well Counter

1~3i-20,000 cpm Rbs-5,000 cpm I:Rb= 1:0.25 YoRecovered:25% Rbs6 Uplake'75%

FIG.6. Double isotope procedure for estimation of myocardial Rb" extraction.

a constant rate from a carotid artery into the circumflex and anterior descending branches of the left coronary artery. Perfusion pressure was recorded continuously to indicate changes in vascular resistance (VR) . Blood was drawn continuously from the coronary sinus via a small catheter and passed through a well counter for radioactivity measurement and a densitometer for O2 saturation measurement and returned to a jugular vein. Il3l-alburnin and Rbs6 in the same syringe were injected rapidly into the arterial inflow and a sample of the coronary sinus blood was collected a t the peak of radioactivity. Comparison of the ratio of I131:RbsG injected to the same ratio in the sample of sinus blood permits computation of E a8 follows:

DEVELOPMENT OF ANTIANGINAL DRUGS

E=

(F)- (F)

(3

or 1 -

71

(&)(%)

where Ii and Rbi are counts injected and IT and Rbr are counts recovered in the sample of sinus blood. Since the rate of coronary flow is constant, changes in C (effective capillary flow) are the same as those in E. Drugs were administered into the arterial inflow (intracoronary). Rbsa extraction was determined during the height of drug action based on changes in VR and compared with values before and after the effect of the drug. These studies demonstrate that changes in E RbE6are not necessarily related to changes in VR or 0, consumption. Nitrites such as nitroglycerin and pentaerythritol tetranitrate increased E RbEs and decreased VR, suggesting arteriolar dilatation and increased effective capillary flow. Norepinephrine decreased E Rbss even though VR was reduced, suggesting arteriolar dilatation but decreased effective capillary flow. When the rate of coronary blood flow was permitted to change, as during perfusion of the coronary arteries a t a constant pressure, the results were more difficult to analyze. Graded doses of nitroglycerin produced graded increases in coronary blood flow but E Rbsa declined progressively. However, C RbsGincreased in a graded fashion. Under these experimental conditions it is difficult to determine whether or not the increase in C RbE6 produced by nitroglycerin is a result of direct action on the capillary circulation or secondary to the increased blood flow rate. Only if the relationship between coronary flow, E, and C were determined with and without drug present could this question be answered. It is of interest to compare our results using regulated coronary flow with those of Love and Burch (1957a,b, 1959) using unregulated coronary flow. Norepinephrine decreased E Rbsa using either procedure, but when blood flow was permitted to rise, C RbE6increased. When blood flow was constant, C Rbse declined. Although Love and Burch (1959) concluded that norepinephrine did not affect E RbsGother than by changes in total blood flow, it is difficult to reconcile their results with ours, for no change in E Rbss should have been observed a t a constant blood flow. In addition to the ease of analysis of results, there is another reason for evaluating antianginal agents a t a constant blood flow rate, namely, the fact that the blood flow rate in the anginal patient is relatively fixed (Section I). Comparison of effect on E Rbsa and oxygen consumption a t constant coronary flow is of interest. Nitroglycerin and pentaerythritol tetranitrate increase E Rbse but reduce oxygen consumption (Fig. 7) whereas norepinephrine decreases E Rb8" but increases oxygen consumption (Fig. 8 ) .

72

MARTIN M. WINBURY

- 7.2 cor. sinus ' 0 content VOI.

-6.4

Yo

-

'

t

j

Rb'%

.t

Sample

N

1 Flow -68 ml/min

mmHg -5'6 200-

perfusion pressure 100-

Nitroglycerine 4Y

0-

RbS6

Somple

Post Nitroglycerine o/oRb*6 Uptake=81.4%

, ,

Conlrol yo Rbe6 Uptake = 76 9%

30 sec

FIQ.7. Effect of nitroglycerin on coronary vascular resistance, Rb" extraction, and cardiac oxygen consumption. Blood flow into left coronary artery at a constant rate and nitroglycerin injected into flow stream. Rb" and I1a'-albumin injected and 2 content curve is temporally out of sample taken a t arrows. The coronary sinus 0 phase with perfusion pressure curve but arrows indicating injections correspond temporally. Vascular resistance is reflected by perfusion premre. Changes in O2 consumption are the opposite of those in coronary sinus 0,content. Rb" uptake is given at bottom of figure. Note decline in vascular resistance and O2 consumption but increase in E Rbm. Val.% cor. sinus

o2 content

-6.4

VOl..%

mmHa -5.6 perfusion

pressure 100-

-

Flow i68 ml/min

U-

Control YORbasUptake = 80.1yo

Post Norepi,%Rbe6 Uptake '71.7%

~ I O ~ C ,

FIQ.8. Effect of norepinephrine on coronary vascular resistance, RbM extraction, and cardiac oxygen consumption. Legend as in Fig. 7. Note decline in vascular resistance and E Rbm but increase in oxygen consumption.

Nitroglycerin given simultaneously with norepinephrine does not prevent the changes in E RbE6 or oxygen consumption due to norepinephrine. 3. Conclusion

The use of diffusible tracers such as RbE6and K42offers a new approach to the evaluation of antianginal agents, since it is possible to study the effects on capillary or nutritional circulation, Measurement of total blood flow does not provide this type of information. There are many physiological aspects of extraction and clearance of RbE6that must be considered in order to interpret the action of drugs. However, by regula-

DEVELOPMENT OF ANTIANQINAL DBUQS

73

tion of some of these factors, i.e., blood flow rate, i t is possible to devise procedures that will be useful. VI. Other Potential Approaches

I n Section V, established procedures and techniques were discussed from the theoretical and practical standpoint. On further reflection about the physiology and biochemistry of the heart (see Section 11) , i t becomes evident that there are other potential avenues of approach. The objective of this section is to present several of these thoughts with the hope that someone will find them s d c i e n t l y logical to warrant exploration in the future.

A. HEMODYNAMIC 1. Heart Rate and Systolic/Diastolic Ratio

Since the greater portion of the coronary inflow occurs during diastole, it should be possible to increase the rate of coronary flow without vasodilatation merely by a prolongation of diastole. Such a mechanism would be effective even when the coronary vessels are incapable of vasodilatation. If the increase in diastolic duration is accompanied by an increase in the total cycle duration (bradycardia) there would be an additional advantage because a reduction in heart rate will decrease myocardial oxygen requirements. Bradycardia due to an increase in systolic duration could have disadvantages in that the systole/diastole ratio would be increased and the TTI/minute might be greater; these changes could lead to a decreased coronary flow and increased oxygen requirement. Thus, the desired feature is a decreased systolic/diastolic ratio with or without bradycardia and without much change in diastolic tension. 2. Autoregulation

Assuming that the coronary blood flow rate is under the basal control of an autoregulatory mechanism (Section 11, C), part of the problem in angina might be associated with a reduction in the sensitivity of the arterioles (or smaller vessels) to the mediator (adenosine?). An agent which has no direct vascular effect, but which would increase the sensitivity of the arterioles to autoregulation should permit a greater than normal increase in coronary flow in response to myocardial hypoxia. I n effect, this means that the basal coronary flow would be within the normal range, but under a stress such as exercise or catecholamines, which leads to tissue hypoxia, there would be an increased perfusion of the capillary beds.

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MARTIN M. WINBVRY

B. METABOLIC 1. Resting versus Activity Oxygen Consumption

The resting oxygen requirement of the heart in vivo ranges somewhere between 25 and 35% of the total requirement during activity (beating). Catecholamines increase the resting oxygen requirement and the percentage it occupies of the total oxygen requirement. Presumably, the energy released in the resting state is used for the maintenance of cellular integrity, accumulation of ions against a diffusion gradient, and the production of high-energy phosphate and heat. One question that arises is whether or not the high-energy phosphate produced is in excess of that required for the activity period (systole) and may be dissipated in the form of heat. Can any phase of basal metabolism temporarily be suppressed without impairing cellular integrity and contractile function, and can the increase due to norepinephrine be blocked by a /I-adrenergic-blocking agent? I n this way it might be possible to reduce the oxygen requirement of the heart so that hypoxia would not develop under stress. The problem is to find a drug that would specifically affect the noncritical portion of the basal metabolism. Out of the total energy output during activity, heat accounts for 3/4 with mechanical work comprising l/. The heat produced is associated with the mechanical and osmotic work performed, the change in entropy due to energy transformation, and the chemical restitution process. Although most of the heat production is obligatory, one wonders if some could be avoided and thereby reduce energy requirements. If the highenergy phosphate produced in the resting period is in excess of the requirement for activity, is this excess degraded and the energy released as heat without production of useful work? This, of course, is related to the comments made previously about the resting energy production. 2. I n Vivo Study of D P N :DPNH System as Index of Aerobic Metabolism

The DPN:DPNH equilibrium is an indication of the state of cellular oxidative function; when hypoxia occurs the equilibrium shifts toward the reduced form ( D P N H ) . Indirect information about this system can be obtained from the lactate-pyruvate equilibrium, for when aerobic mechanisms are not available for regeneration of D P N (hypoxic conditions), pyruvate is reduced t o lactate. Huckabee (1961) devised the “excess lactate” concept for evaluation of the state of cellular oxidation ; excess lactate production occurs during anaerobic metabolism. Any reduction in the function of the aerobic enzyme systems (hypoxia or cyanide) will lead to excess lactate production.

DEVELOPMENT OF ANTIANGINAL DRUGS

75

Determination of the change in the redox (oxidation-reduction) potential of the lactate-pyruvate system as blood passes through the heart can also be used to determine the state of tissue oxidative function (Gudbjarnason et al., 1962; Stock et al., 1962). With adequate oxygenation, the difference between the redox potential of the coronary sinus blood and arterial blood (A Eh) is positive, indicating active tissue oxidation. During anoxia 4 Eh becomes negative, indicating anaerobic metabolism. In principle, the redox potential gives a measure similar to that of the excess lactate procedure, namely, the balance between aerobic and anaerobic metabolism. These procedures have not been applied to a systematic evaluation of drugs used in the treatment of angina. Such a study might be enlightening and offer a new approach to the evaluation of antianginal drugs. 3. Direct Intracellular Visualization of Oxidation-Reduction Status

Chance et al. (1962) has described a procedure which permits estimation of changes in the DPN:DPNH system of the mitochondria of intact cells by measurement of the emission spectra from an area 20 p in diameter on the surface of an organ. The excitation wavelength was 366 mp and the emission wavelength 472 mp. There was an increase in the intensity of emission in going from an aerobic to anoxic state ( D P N reduction). This procedure has been applied to the kidney and brain and i t was found that anoxia or hydrogen sulfide produced an abrupt increase in fluorescence, indicating an increase in pyridine nucleotide reduction. This procedure should permit study of the intracellular metabolic state of the heart and the effect of drugs thereon. 4. Stress-Adaptation Mechanism

During extreme cardiac work loads, tachycardia, or hypoxia a sudden reduction in myocardial oxygen consumption occurs even though the cardiac work level, tachycardia, or hypoxia is maintained (Kate, 1956; Kate et al., 1955; Laurent et al., 1956). This provides a means by which the heart can adjust to sudden and excessive stress. It has been suggested that the mechanism involves the sudden release of anaerobic energy (Ballinger and Vollenweider, 1962). Does a similar mechanism appear during an acute anginal episode or during the relief thereof by an effective nitrite? If a drug were able to induce such a change i t should prevent or reduce coronary insufficiency induced by stress. Perhaps what is required is an agent that would lower the threshold of stress required to initiate the “stress-adaptation” mechanism.

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MARTIN M. WINBURY

5. Eficiency Efficiency can be considered from two points: (1) mechanical efficiency, which is in terms of work output in relation to oxygen consumption, and (2) biochemical efficiency, which is in terms of high-energy phosphate produced in relation to oxygen consumed. Since ( 1 ) is a measure of the over-all efficiency of the system it is, in part, determined by (2). It has been estimated that the maximal possible biochemical efficiency for the oxidation of glucose is 39% (Bing and Michal, 1959). This is based on the development of 266 kcal of high-energy phosphate (38 moles X7000 cal/mole) for the 686 kcal of free energy liberated on complete oxidation of a mole of glucose. It has not been possible to determine the level of biochemical efficiency in the intact heart, and the actual value may well be below 39%. Under normal circumstances the biochemical efficiency may be the same in the heart of the normal and anginal patient, but under stress, such as exercise, differences may appear. I n the normal heart, acceleration of metabolism may cause no change or a rise in biochemical efficiency, whereas, in the anginal heart, the ability to accelerate metabolism may be limited, or the biochemical efficiency may decline. This could be a result of a limited capacity of some phase of the metabolic machinery involved in the conversion of substrate to high-energy phosphate. Thus, tissue hypoxia would result even though an adequate oxygen supply were present. Certainly the results in man, which show a rise in over-all mechanical efficiency of the heart in normal individuals during exercise but a decline in anginal patients, are in agreement with such a concept. It is difficult to conceive of an improvement in the mechanical efficiency other than by hemodynamic changes (Section 11,C) or by alterations in the biochemical efficiency. It is true that the calculated mechanical efficiency is increased as the work level rises because the resting oxygen requirement remains relatively constant, and only the activity requirement increases; but this is not a real improvement in energy transfer. However, one does not know if the TTI/Qo, ratio could be improved by coupling a greater amount of the phosphate bond energy to useful work. REFERENCES Alella, A., Williams, F. L., Bolene-Williams, C., and Katz, L. N. (1955). Am. J. Physiol. 183, 570. Asada, S., Chiba, T., Osawa, K., Nakamura, K., and Murakawa, S. (1962). Japan. Circulation J. [English Ed.] 28, 849. Ballard, F. B., Danforth, W. H., Naegle, S., and Bing, R. J. (1960). J . Clin. Invest.

39, 717. Ballinger, W. F., and Vollenweider, H. (1962). Circulation Ree. 11,681.

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Bennish, A., and Bing, R. J. (1962). J. Lab. Clin. Med. So, 859. Berglund, E., Monroe, R. G., and Schreiner, G. L. (1957). Acta Physiol. Scand. 41, 261.

Berne, R. M. (1958). Circulation Rea. S, 644. Berne, R. M. (1963). A m . J. Physiol. 204,317. Berne, R. M., Blackmon, J. R., and Gardner, T. H. (1957). J . Clin. Invest. 36, 1101. Beuren, A., Sparks, C., and Bing, R. J. (1958). Am. J. Cardwl. 1,103. Bing, R. J. (1959). Ann. N . Y . Acad. Sci. SOy 1030. Bing, R. J. (1961). Acm. J. Med. 30, 679. Bing, R. J., and Michal, G. (1959). Ann. N . Y . Acad. Sci. 72, 555. Bing, R. J., Choudhury, J. D., Michal, G., and Kako, K. (1958). Ann. Internal Med. 49, 1201.

Bing, R. J., Danforth, W. H., and Ballard, F. B. (1960). J . Am. Med. Assoc. 172, 438. Black, J. W., and Stephenson, J. S. (1962). Lancet 11, 311. Blumgart, H. L., Levine, S. A., and Berlin, D. D. (1933). Arch. Internal Med. 51,868. Blumgart, H. L., Freedberg, A. S., and Kurland, G. 9. (1951). New Engl. J . Med. 245, 83.

Brachfeld, N., Bozer, J., Gorlin, R. (1959). Circulation 19, 697. Braunwald, E., Sarnoff, S. J., Case, R. B., Stainsby, W. N., and Welch, G. H., Jr. (1958). A,m. J . Physiol. 192, 157. Bretschneider, H. J., Frank, A., Bernard, U., Kochsiek, K., and Scheler, F. (1959). ArzneimitteGForsch. 9, 49. Cahn, J., and Herold, M. M. (1962). Chemotherapia 4, 262. Calesnick, B. (1963). Angiology 14, 17. Carlsten, A., Hallgren, B., Jagenburg, R., Svanborg, A., and Werko, L. (1962). Metab. Clin. Exptl. 11, 814. Cesarman, T. (1959). Ann. N . Y . Acad. Sci. SO7 988. Chance, B., Cohen, P., Jobsis, F., and Schoener, B. (1962). Science 137, 499. Chappel, C. I., Rona, G., Balazs, T., and Gaudy, R. (1959). Arch. Intern. Pharmacodyz. 122, 123. Charlier, R. (1961). “Coronary Vasodilators.” Pergamon Press, New York. Chidsey, C. A., Harrison, D. C., and Braunwald, E. (1962). New Engl. J . Med. 267,

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Conn, H. L., Jr. (1962). Circulation Res. 10, 505. Conn, H. L., Jr., Wood, J. C.,and Morales, G. S. (1959). Circulation Res. 7, 721. Cossio, P. (1959). Ann. N . Y . Acad. Sci. SO, 1009. Coulshed, N. (1960). BTit. Heart J . 22, 79. Cross, C. E., and Oblath, R. W. (1962). A m . J. Physiol. 202,616. Crumpton, C. W., Castillo, C. A,, Rowe, G. G., and Maxwell, G. M . (1959). Ann. N . Y . Acad. Sci. SO7 960. Danforth, W. H., Naegle, S., and Bing, R. J. (1960). Circulation Res. 8, 965. Darby, T. D., and Aldinger, E. E. (1960). Circulation Res. ‘8,100. Darby, T. D., and Gebel, P. P. (1962). Pharmacologist 4, 180. Darby, T. D., Sprouse, J. H., and Walton, R. P. (1958). J. Pharmucol. Ezptl. Therap. 125 386. DeGraff, A. C., and Lyon, A. F. (1963). A m . Heart J . 69,423. Denison, A. B., Jr., and Green, H. D. (1958). Circulation Re&.6,833. Denison, A. B., Jr., Bardhanabaedya, S., and Green, H. D. (1966). Circulation Rea. 4, 653. DiPalma, J. R. (1961). Angwlogy 1%564.

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Pharmacological Aspects of Parkinsonism ALEXANDER H. FRIEDMAN Department of Pharmacology and Toxicology, University of Wisconsin Medical School, Madison, Wisconsin AND

GUY M. EVERETT Section of Neuropharmacology, Abbott Laboratories, North Chicago, Illinois; Department of Pharmacology and Therapeutics, Stritch School of Medicine, Loyola University, Chicago, Illinois

I. Introduction . . . . . . . . . . . . 11. Nature of the Disease . . . . . . . . . Pathophysiology of Parkinson’s Disease . . . . . 111. Conventional Pharmacotherapy of Parkinsonism . . . IV. Histamine and Antihistamines . . . . . . . V. Screening Methods for Anti-Parkinson Drugs . . . . A. Bioassay, Nicotine Tremor, and Surgically Induced Tremor B. Harmine Tremor . . . . . . . . . . C. Tremorine Tremor . . . . . . . . . D. Cholinergic-Blocking Activity . . . . . . . E. Miscellaneous Methods . . . . . . . . VI. Drug-Induced Parkinsonism . . . . . . . . A. Rauwolfia Alkaloids . . . . . . . . . B. Phenothiazine Derivatives . . . . . . . C. Butyrophenones . . . . . . . . . . VII. Rational Pharmacotherapy . . . . . . . . VIII. Summary . . . . . . . . . . . . References . . . . . . . . . . . .

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I. Introduction

The pharmacotherapy of Parkinsonism justifiably is undergoing important changes. The empirical approach of the past to the control of this condition has been only partly successful in solving the attendant problems, while the present orientation predicts rational medication because the underlying bases of the disease are more fully appreciated. It is the aim of this chapter to define the conditions known as Parkinson’s disease and to examine the pertinent pathophysiology, especially with regard to 83

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the fusimotor system; to outline common approaches t o drug therapy; to examine the methodology employed for screening and designing new drugs, particularly the effects of Parkinsonimimetic or tremor-producing agents as they might contribute to the understanding of Parkinsonism; and to review briefly the nature of drug-induced Parkinsonism for the insights it might furnish in determining a rational approach to the pharmacotherapy of Parkinson’s disease. II. Nature of the Disease

The shaking palsy (idiopathic Parkinsonism), first described in 1817 by James Parkinson, presents a picture of involuntary tremor, muscular rigidity, weakness, and akinesia, masked facies or “diplomat’s expression,” bent posture, characteristic “pill-rolling” motion of the hands, and a festinating gait. The senses and the intellect are not impaired, although the patient may be mentally depressed as a reaction to his illness. The age of the patient at the onset of this progressive disease is 45 to 60 years. The average age of onset has shown a tendency to increase along with that of the general population. Modern chemotherapy has markedly curtailed the postencephalitic form often found in patients under 40, and the older segment of the population affected by influenza encephalitis following World War I is rapidly dying off. Specific findings in postencephalitic Parkinsonism, in addition t o those mentioned, may also include oculogyria, convergence insufficiency, pupil abnormalities, sialorrhea, excessive perspiration, and seborrhea. I n arteriosclerotic Parkinsonism, which affects those over 60, classic signs of the disease are mild, and rigidity is the predominating feature.

PATHOPHYSIOLOGY OF PARKINSON’S DISEASE Insight into some of the underlying mechanisms of Parkinson’s disease might be obtained by examining the various surgical techniques that have evolved over the last 70 years for the alleviation of symptoms. Excision of the precentral cortex for the relief of athetosis was first tried in 1890 by Horsely (1909). Klemme (1940) and Bucy (1949) advocated that lesions of the pyramidal tract produced by excision of premotor and motor cerebral cortex would be of value in various motor disorders since this pathway ultimately innervates the anterior horn cells. Palliation of tremor but not rigidity was achieved in some of these cases, but relief was obtained a t the expense of motor movement. I n addition, the lesions gave rise to convulsive episodes, and the mortality rate was high. Disruption of the pyramidal tract a t the level of the cerebral peduncle was utilized by Walker (1952) for the relief of tremor and rigidity (sometimes a t the

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cost of hemiparesis or hemiplegia). Putnum (1942) devised the pyramidotomy for this purpose, placing lesions a t the level of the second cervical segment of the spinal cord. Tremor was relieved ipsilaterally a t the expense of motor power without the dangers connected with cortical excision. Oliver (1953), using a modification of Putnum’s technique, concluded that unilateral tremor is the only indication for pyramidotomy. This in essence appears to be the scope of pyramidal tract lesions. They relieve tremor a t the expense of motor power but have little effect on other equally distressing symptoms of Parkinsonism. Partial relief of rigidity has been obtained by dorsal root section (Pollock and Davis, 1929) or by the injection of local anesthetics (Walshe, 1924), but results have not been especially impressive. Risteen and Volpitto (1946) noted that stellate ganglion blockade reduced the spasticity resulting from cerebrovascular accidents. They attributed the improvement to the interruption of vasoconstrictor impulses to the brain. This procedure appeared to be somewhat effective against Parkinsonian rigidity, but did not alter tremor. Gardner (1949) found that procaine blockade of the cervical sympathetic chain or sympathectomy afforded transient relief which was so slight as to be of little therapeutic value in Parkinsonism. A major advance in the surgical therapy of Parkinsonism was initiated by the studies of Russell Meyers on the basal ganglia (1940, 1951). It has subsequently been established that lesions of the globus pallidus, ansa and fasciculus lenticularis, ventrolateral nucleus of the thalamus, substantia nigra, or midbrain tegmentum may be beneficial in alleviating rigidity and resting tremor. It is also clear that the caudate nucleus and putamen are not suitable sites for this purpose (Cooper, 1961). Rand et al. (1962) have shown that in man the greatest motor activity following stimulation of the brain occurred in the region of the ventralis, lateralis, and centrum medianum of the thalamus and suggested this, in agreement with Hassler (1955), as the best surgical target for the therapy of resting and action tremor. The surgical target of choice for the relief of both tremor and rigidity is, according to Cooper (1961), the region of the ventrolateral nucleus of the thalamus, a region whose primary blood supply is via the anterior choroidal artery, and which receives impulses from the globus pallidus, red nucleus, vestibular nucleus, and the cerebellum. This area exerts important influence on the cerebral cortex and pyramidal tract. Henner (1962) has pointed out the antithetic relationship between cerebellar deficit and the Parkinsonian syndrome. It is apparent that common pathways are involved in these hyperkinetic states, since interruption of fibers from the dentate nucleus of the cerebellum to the thalamus, either via the dentatothalamic tract or less directly via the dentatorubral and rubrothalamic tracts, is capable of abolishing resting

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or intention tremor. Utilizing the action of ethanol, Henner has demonstrated suppression of the motor symptoms of Parkinson patients followed by the appearance of signs of cerebellar deficit as the dose was augmented. He has also shown that bulbocapnine can temporarily ameliorate cerebellar deficit, but produces Parkinsonian-like reactions with increasing doses. Cooper, reviewing his experience of more than 1500 operations (1961) has found that “a properly placed lesion” in the mesial globus pallidus relieves rigidity in 75% and tremor in 60% of “properly selected cases.” Tremor and rigidity are relieved by a properly placed lesion in the ventrolateral nucleus of the thalamus in “90% of subjects who are selected with care and in whom the technique is meticulously performed.” Unfortunately, despite the optimism which might be engendered by such a report, neurosurgery does not alleviate other problems which are a part of the Parkinson syndrome, such as dysarthria, dysphagia, sialorrhea, and motor defects associated with posture and gait as well as with fine movement (England and Schwab, 1961b). These in themselves are perhaps as distressing and debilitating to the patient as those that might be relieved by neurosurgery. Added to these considerations must be the attendant uncertainty and irreversibility of outcome. Thus, neurosurgery can be considered to be only a very partial solution to the problem of Parkinsonism. Also to be considered in the pathophysiology of Parkinsonism are certain peripheral regulating mechanisms that modulate motor activity. Particular attention is drawn to the role of proprioception and the fusimotor system. While a great deal of emphasis has been placed on corticoor subcorticospinal regulation of anterior horn cell activity (Bucy, 1949; Meyers, 1942; Ward et al., 1948; Spiegel and Wycis, 1958; Adey e t al., 1960; Carpenter et d., 1957; Benda and Cobb, 1942; Denny-Brown, 1960 a,b), interest in the role of the fusimotor system (formerly designated the y or small motor system) in the regulation of motor activity has increased over the past decade. Briefly, the muscle spindle, which is oriented parallel to and within the extrafusal or main muscle mass, is a length sensor of a sensitive proprioceptive servo system which acts to regulate the length of the main muscle mass. Excitation of the spindles’ equatorial sensory apparatus-contained within the nuclear bag-feeds back information to the spinal cord which modulates impulses directed to the muscle. Because of the parallel arrangement, contraction of the extrafusal muscle by the direct or a-route “unloads” the spindle and reduces the firing frequency or even silences the sensory element. The yroute conducts impulses to the contractile poles of the muscle spindle, the stretch causing the sensory portions to fire a t an increasing rate until the extrafusal muscles shorten sufficiently to offset the increase. This indirect

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mechanism has been termed the “follow-up length servo” (Hammond et al., 1956). The functioning of the fusimotor system is complicated by the interaction of other feed-back mechanisms such as the Renshaw collaterals, which behave as a frequency modulating monitor of anterior horn cell activity (Granit, 1959; Struppler e t al., 1960), or the Golgi tendon organs, which transmit inhibitory impulses to the spinal cord (Asai and Hufschmidt, 1958; Hufschmidt, 1960). There is general agreement that, in the production of Parkinson tremor and rigidity, the activity of the fusimotor system is altered. It is the nature of the alteration which is a source of controversy. On the one hand, Parkinsonism is attributed in part to hypoactivity of the y-loop (Hassler, 1955; Stern and Ward, 1962; Jung and Hassler, 1960), while on the other, this servo system is considered to be hyperactive (Rushworth, 1960; Barraquer-Bordas, 1958). In classic Sherringtonian decerebrate rigidity, y-efferent activity is augmented (Eldred et al., 1953; Matthews and Rushworth, 1958), whereas in the rigidity produced by cerebellar ischemia (Pollock and Davis, 1929), the activity of the a-motor neurons is increased (Tereuolo and Tereian, 1953). Deafferentation by posterior rhieotomy (Pollock and Davis, 1930) or by local anesthesia (Liljestrand and Magnus, 1919; Walshe, 1924; Matthews and Rushworth, 1957; Matthews, 1958) relieves the former type of rigidity but does not affect the latter. Deafferentation also relieves Parkinsonian rigidity but worsens tremor by increasing its amplitude (Walshe, 1924; Pollock and Davis, 1930), indicating that the peripheral length servo plays a role in regulating tremor. However, the regulation of frequency apparently is a function of the central nervous system (Stern and Ward, 1960; Li, 1956; Eldred et al., 1953; Granit and Kaada, 1952). The evidence for decreased activity of the spindle servo mechanism rests partly on the failure of the Jendrassik maneuver to facilitate the tendon reflex in Parkinsonian patients (Hassler, 1955; Jung and Hassler, 1960). Hoffmann (1962a; Hoffmann et al., 1962), however, has found that the fusimotor system is available to y-facilitation, and attributes Parkinsonian rigidity to defective bias on the muscle spindle, As a consequence, simultaneous unimpeded bombardment of the a-motor neurons of agonist-antagonist pairs occurs. Failure of central regulating mechanisms in Parkinson patients may lead to concurrent firing of both a- and 7-neurons a t a-velocity (Granit et al., 1959; Rutledge and Haase, 1961). With regard to the mechanism of tremor, Hoffmann (1962b) has found that a profound afferent silence follows a tremor burst, suggesting that the periodic a-firing is accompanied by heavy 7-efferent driving. This accelerates facilitatory and inhibitory impulses to agonist and antagonist, developing the extended spindle pause when extrafusal firing exceeds intrafusal firing. This is very interesting in view of the preliminary findings of one of us (A.H.F.) that

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ALEXANDER H. FRIEDMAN AND GUY M. EVERETT

tremorine (1,4-dipyrrolidino-2-butyne) in the dog lengthens the silent period produced by a stimulus applied to the a-motor fiber. Furthermore, studies in the cat, also preliminary, indicate that 7-efferent activity (as monitored by spindle afferents) is augmented by tremorine. Henatsch and Ingvar (1956) have shown that chlorpromazine influences fusimotor activity by depressing y-efferent activity. They were able to abolish intercollicular decerebrate rigidity but not the rigidity of cerebellar ischemia with this drug. This finding suggests a t least one possible explanation of the action of phenothiazine derivatives in producing extrapyramidal reactions. Stern and Ward (1962) found that chlorpromazine greatly augmented a-motor discharge in an intact a-preparation and postulated from this observation and from other extensive studies that Parkinsonian tremor, rigidity, and akinesia resulted from “an alteration of the normal alpha-gamma balance, with a relative depression of gamma activity and heightened alpha activity.” The central actions of chlorpromazine have recently been reviewed by Dasgupta (1961),who, with Werner (Dasgupta and Werner, 1955), studied the inhibitory actions of chlorpromazine on motor activity. Himwich and Rinaldi (1957) demonstrated its depression of the reticular formation and diffuse thalamic projections that comprise the mesodiencephalic system. The action of chlorpromazine and other phenothiazines on the brain-stem reticular formation is discussed in a recent review of drug action in this area by Killam (1962). Chin and Smith (1962) have shown that caramiphen hydrochloride (Panparnit), which has enjoyed use as an anti-Parkinson drug, decreases fusimotor discharges, thus relieving rigidity in the decerebrate cat. C. M. Smith (1960) has also shown that large doses of atropine in vitro will decrease the afferent discharge of the frog muscle spindle, although he was unable to demonstrate this action in the cat in vivo after the administration of 2 mg/kg intravenously, This apparent discrepancy is of interest in view of Hunt’s (1952)finding that acetylcholine will augment spindle discharge in the cat. It might be that the dose level used in the cat was too small, or that the local anesthetic action of atropine became apparent at the high doses used in the in vitro study. C. M. Smith (1963) in an invaluable review has considered the direct and indirect actions of a variety of drugs on the fusimotor system. Several of the studies reviewed indicate that the fusimotor system of the Parkinsonian patient differs in its behavior from that seen in normal individuals. Mention should be made of Byrne’s (1926) finding of degeneration of the intrafusal nerve and spindle in postencephalitic Parkinson patients. No other reference to fusimotor degeneration in Parkinsonism had appeared in the literature to our knowledge until the recent report of Tuncbay and co-workers (1961) who found striking abnormalities of both

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intra- and extrafusal motor nerves in their series of 8 patients, although the sensory fibers of the spindle organ were found to be normal. It is unclear whether the observed degeneration is of a primary or secondary nature, but it is in keeping with the interpretation of defective fusimotor activity in Parkinsonism. I l l . Conventional Pharmacotherapy of Parkinsonism

The physician’s armamentarium for the treatment of Parkinson’s disease is extensive, which is indicative of the status of our present knowledge of the underlying mechanism of this condition. The following tables, modified from Doshay (1961), summarize concisely much of what is regarded as conventional pharmacotherapy of Parkinsonism. Symptomatic relief rather than cure is achieved by these means. The early drugs, the effectiveness of which was determined empirically, were alkaloids derived from such solanaceous plants as Atropa belladonna, Hyocyumus niger, and Datura strumonium, and are primarily cholinergic-blocking agents both centrally and peripherally, although they also possess other pharmacological properties. Atropine, for example, has half the relative local anesthetic potency of procaine (De Elio, 1948) as well as some antihistaminic activity (Loew et ul., 1946; Dutta, 1948). I n an attempt to circumvent the troublesome toxic manifestations of these drugs (e.g., xerostomia, blurred vision, and a tendency to glaucoma) and tolerance to continued use, synthetic compounds were developed. Table I lists standard anti-Parkinson drugs. Trihexyphenidyl, cycrimine, and procyclidine are designated as “universal” in their action, i.e., they ameliorate to some extent all signs of Parkinsonism (Doshay et al., 1954; Zier and Doshay, 1954, 1957). Biperiden, described in the N.N.D. (A.M.A. Council on Drugs) for the first time in 1961, is comparable to this group in its action. The remaining drugs listed are termed “specific” because they are relatively selective in their action against a particular feature of Parkinson’s disease ; thus benatropine methanesulfonate is of value in the treatment of rigidity (Doshay, 1956), orphenadrine for akinesia (Doshay and Constable, 1957), as is phenoxene (Doshay and Constable, 1959), and ethopropaaine for the treatment of tremor (Doshay et al., 1956). The “specific” agents are often less than specific in their pharmacological actions, All drugs listed have some antihistaminic (for further discussion vide infru) and cholinolytic activity. Benatropine, in addition, exerts local anesthetic action and ethopropazine produces ganglioplegia. It is apparent from the above that the effectiveness of these drugs cannot be explained easily on the basis of any one or combination of these actions. “Universal” drugs are frequently used in combination with “specific” drugs for improved pharmacotherapy, e.g., trihexyphenidyl with

(0

0

STANDARD

Generic name

Trade name

Trihexyphenidyl (benzhexol)

Artane

Cycrimine

Pagitane

Procyclidine

Kemadrin

Biperiden Benztropine methanesulfonate

Akineton Cogentin

Orphenadrine

Disipal

Chlorphenoxamine

Phenoxene

Ethopropazine

Parsidol

a

Modified from Doshay (1961).

TABLE I DRUGSFOR PARKINSON'S DISEASE'

Indications Basic drug for all symptoms

Side effects

Dry mouth, blurred vision. Overdosage causes confusion, delirium, and hallucinations Same as trihexyphenidyl Fewer reactions than with large doses of trihexyphenidyl Same as trihexyphenidyl Fewer reactions than with large doses of trihexyphenidyl Same aa trihexyphenidyl Atropine-like, mental confusion Muscle s p a m , cramps, severe Dryness of mouth, occasional rigidity, contractures, skin reaction frozen states Fatigue, weakness, sluggishSlight dryness of mouth ness, depreeaion, sialorrhea diaphoresis, rigidity Same aa orphenadrine, but Slight dryness of mouth longer lasting Good for tremor control, if Drowsiness, dizziness tolerated. Good muscle relaxant

Manufacturer Lederle

Burroughs Wellcome Knoll Merck Sharp & Dohme Riker Pitman-Moore Warner-Chilcott

TABLE I1 SUPPLEMENTARY DRUGS FOE PARKINSON'S DISEASE" Generic name

Trade name

&Amphetamine

Dexedrine

Amphetamine

Benzedrine

Desoxyn Deoxyephedrine Diphenhydramine Benadryl Phenindamine Promethazine

Thephorin Phenergan

Chlorpromazine Meprobamate Barbiturates

Thorazine Equanil; Miltown Many

Zoxazolamine Hyoscine

Flexin Many

Rauwolfia alkaloids

Many

Belladonna alkaloids Imipramine

Rabellon; Bellabulgara Tofranil

a

Modified from Doshay (1961).

Indications

Side effects

Lethargy, akinesia, oculogyric Excitations, restlessness, palpitations, increase in tremor crises More severe than Dexedrine, Same as &hetamine especially cardiac effects Simiiar to &hetamine Same aa &hetamine Drowsiness, dizziness Tension, excitement, tremor, and insomnia, if tolerated None apparent Hypersensitive patients Dryness, occasional indigestion Slight muscle relaxation, antihistaminic None, if used only at bedtime Insomnia Tranquilizer for nervousness, Drowsiness, dizziness, mental fogginess, occasional indigestion restlessness, insomnia Drowsineea, possible habituation, Nervousness, restlessness, slowing down of motor function insomnia Slight muscle relaxant action Weakness, sluggishness Severe dryness of mouth, blurring Good for tremor control, if of vision tolerated Stuffy nose, loose bowels, Nervousness, restlessness, drowsiness high blood pressure Muscle relaxation Akinesia, mental depression

Slight dryness of mouth, blurring of vision Sweating, dryness of mouth

Manufacturer Smith, Kline & French Smith, Kline & French Abbott Parke, Davis Hoff man-LaRoche Wyeth Smith, Kline & French Wyeth; Wallace All manufacturers McNeil

AU manufacturers Large variety of products from different manufacturers Merck Sharp & Dohme; Harbor Geigy

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ALEXANDEB H. FRIEDMAN AND GUY M. EVERETT

ethopropazine (England and Schwab, 1961a), and ethopropaeine with orphenadrine ( Gillhespy, 1960). Table I1 includes a variety of supplemental drugs that are used conjointly with those listed in Table I. The list is by no means complete, but indicates to some extent the nature of ancillary therapy. Central stimulants such as &hetamine are intended to relieve lethargy, akinesia, and oculogyria. However, they may increase tremor (Doshay, 1958). Other stimulants such as methylphenidate, pipradol, and even pentylenetetrazole, intended to diminish apathy and elevate the mood, have been of little value. On the other hand, imipramine, a drug used in mental depression, lessens akinesia and appears to limit the emotional exacerbation of tremor and rigidity and is well tolerated by the patient (Mandell et al., 1961). Internuncial depressants such as meprobamate, mephenesin, zoxaeolamine, and methocarbamol may relieve muscle spasm and rigidity in some patients, but in general are not considered useful in Parkinsonism. Barbiturates may sedate the patient for a restful night of sleep, but if they are long-acting they may increase his motor deficit. Summarizing their view of current pharmacotherapy of Parkinsonism, England and Schwab (1961a) state that “with the best use of all these agents in optimal adjusted dose and combination, the objective improvement is no more than 25 percent and may be closer to 10 percent, which, of course, is disappointing.” However, according to Doshay (1961), when pharmacotherapy is initiated early enough in the properly motivated patient and combined with intensive physiotherapy, sialorrhea, oculogyria, diaphoresis, insomnia, dysarthria, akinesia, rigidity, and tremor can be ameliorated in a majority of cases. IV. Histamine and Antihistamines

Antihistamines are used effectively in the treatment of true Parkinson’s disease (Budnitz, 1948; McGavack et al., 1947) and of drug-induced Parkinsonism (M. J. Smith and Miller, 1961; Flegenheimer, 1959; Waugh and Metts, 1960; Marcus et al., 1960; P. L. McGeer et al., 1961). Diphenhydramine was first used for this purpose on an empirical basis. Code (1945) and Harris et al. (1946) described the cholinergic-blocking action of diphenhydramine (Benadryl). In the following year, McGavack e t al. (1947) used this drug to treat 4 cases of paralysis agitans. Improvement was noted in 3 and was attributed in part to augmented circulation to the corpus striatum resulting from congestion induced by this drug in the choroid plexus. Does histamine play a role in the etiology of Parkinsonism? Studies relating to the central action of this substance are scarce. Ungar and Witten (1963) demonstrated that, following the administration of tremorine in fairly high doses to rat and dog, histamine in the brain rose to about twice its normal concentration. The content of the corpus

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striatum of the dog, for example, increased from 0.156 pg/gm wet weight to 0.331 pg/gm or 212%. This effect was inhibited by atropine. Mescaline and chlorpromazine slightly elevated rat brain histamine, while reserpine, lysergic acid diethylamide (LSD),strychnine, amphetamine, and Metrazol were without effect. The significance of these findings is unclear. Button (1953) has utilized the intravenous administration of histamine for the treatment of Parkinsonism for a number of years with some success. No physiological basis for the sense of improvement in his patients is offered, but it is suggested that an augmented cerebral blood flow may contribute to this effect. Histamine infusion alone does not alleviate Parkinsonism but appears to potentiate the action of more conventional anti-Parkinson drugs. The ubiquity of histamine suggests that it might have some fundamental physiological role, but a direct neurological action seems unlikely in view of the essentially negative results obtained from studies designed to demonstrate such activity (Sick, 1962). A modulator action for histamine, however, cannot be ruled out. The increase in brain-stem histamine following the administration of tremorine (Ungar and Witten, 1963) may be the result of metabolic interference with the degradation of histamine by the inhibition of histaminase (diamine oxidase) or more simply perhaps by a general depression of metabolism, since tremorine does produce hypothermia. On the other hand, a stimulating action on histidine decarboxylase might increase the rate of histamine synthesis and cause the amine to pile up. If histamine has a hitherto unsuspected neurological role in the production of Parkinsonism, it might help to explain the therapeutic efficacy of antihistamines in true and drug-induced Parkinsonism. Antihistamines, in addition to their well-known sedative effects, are capable, in high doses, of stimulating the central nervous system (Gilmore and Athreya, 1960). It should also be pointed out that antihistamines can cause the release of histamines (Paton, 1957). The enhanced histaminase activity after the administration of histamine (Karhdy, 1936) may also help to explain why infusions of histamine may be useful in Parkinsonism. Finally, a fact that bears reiteration is that the antihistamines are only relatively selective in their action, so that their anti-Parkinson activity may also be a reflection of local anesthetic (Dutta, 1948) and/or cholinergic-blocking activity (Reuse, 1948). V. Screening Methods for Anti-Parkinson Drugs

A. BIOASSAY, NICOTINE TREMOR, AND STJRGICALLY INDUCED TREMOR

Various methods are available for the screening of anti-Parkinson drugs [the reader is referred to the recent critical reviews of Vernier (1963) and Everett (1964) 1. Fundamentally these techniques differ with

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ALEXANDER H. FRIEDMAN AND GUY M. EVERETT

respect to their specificity and predictability, i.e., the reliability with which the findings may be extrapolated to man, who ultimately is the final test object for these potential drugs. Methods range from the relatively simple to the complex. The earliest approach was empirical : the bioassay for cholinergic-blocking action. The observation of hyperactivity of the parasympathetic nervous system in Parkinson patients suggested the use of the belladonna alkaloids (Charcot, 1874) and afforded the first effective treatment of paralysis agitans. Human bioassay is still being utilized, but today it is considerably more sophisticated (Nodine et al., 1962) in that it yields dose-response information with respect to clinical improvement and toxicity. Screening procedures in animals have included the technique of Bovet and Longo (1951) in which a transient tremor is produced by the intravenous administration of nicotine. The chief objection to this technique is that it fails to reproduce the features of Parkinsonism and, further, that drugs of proved efficacy in the treatment of this syndrome do not antagonize nicotine tremor (Cahen and Lynes, 1951). Parkinson-like postural tremor has been obtained in monkeys by lesions in the midbrain tegmentum (Ward et al., 1948; Peterson et al., 1949). Jenkner and Ward (1953) among others, have produced tremor in this species by stimulating this area. Spiegel et al. (1960) recently localized the tremorigenic site in man and showed that stimulation caudal to the oral mesencephalic tegmentum produced tremor or amplified an existing one. The difficult technique of placing electrolytic lesions in the midbrain of the monkey has been utilized by Vernier and Unna (1956, 1963) as an assay procedure. This is a relatively costly method since only a few of the operated animals are suitable test objects. In addition, i t is a time-consuming procedure. The assay procedure relies on careful observation and tabulation of tremor activity of the restrained monkey before and after the test drug. While the technique permits measurement of peripheral effects of drugs being studied, it does not consider other motor aspects of Parkinsonism. However, results are reproducible and highly specific, permitting accurate determination of a relative potency. Finally, it corroborates the relative effectiveness of common forms of anti-Parkinson pharmacotherapy.

B. HARMINE TREMOR For more than a century the harmala alkaloids isolatcd from Banisteria caapi and Peganum harmala have been recognized for their marked effects on the central nervous system (Gunn, 1935; A. L. Chen and Chen, 1939). They are known to produce hallucinatory episodes in man and to produce tremor, occasionally rigidity, and frequently convulsions in a variety of species including primates, Harmine and harmaline have been

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used for the treatment of postencephalitic Parkinsonism (Lewin and Schuster, 1929; Halpern, 1930a,b; Astley Cooper and Gunn, 1931) and investigated for their extrapyramidal activity (Hara and Kawamori, 1954). Zetler (1957) conducted an extensive investigation of the antagonism of harmine-induced tremor in mice, since this relatively long-lasting coarse tremor seemed to offer a better test situation for the evaluation of antiParkinson agents than did nicotine tremor. He found, in general, that the phenothiazine tranquilizers were more potent harmine antagonists than were the anti-Parkinson drugs. Their sedative effect and cholinergic blocking activity could not be correlated with the antagonism to harmine tremor although the combination of adrenolytic, sedative, and serotoninolytic action seemed to provide the greatest protection. Among the 41 compounds tested the most potent antagonists were LSD, serotonin, chlorpromazine, promethazine, and apomorphine. Only a small difference in potency was obtained between atropine and scopolamine, although the latter is 10 times more potent in its capacity to depress the midbrain reticular formation (Longo, 1956). On the other hand, benzhexol and caramiphen, which are less potent in their depressant action on the reticular formation (Vernier and Unna, 1963), were considerably more active as harmine antagonists than were the belladonna alkaloids. From these considerations it is clear that as a screening method for anti-Parkinson drugs the blockade of harmine-induced tremor cannot be considered appropriate.

C.

TREMORINE

TREMOR

Tremorine, 1,4-dipyrrolidino-2-butyne,is an especially useful pharmacological tool for the evaluation of anti-Parkinson drugs (Fig. 1 ) . Its capacity to mimic many features of Parkinson’s disease has convinced a number of investigators of its potential value for providing insight -C&-CEC-C&-N

3

Tremorine

CZC-CE&-N

3

Oxotremorine Fro. 1. Structures of tremorine and oxotremorine.

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ALEXANDER H. FRIEDMAN AND GUY M. EVERETT

into the basic mechanism of extrapyramidal tremor and rigidity. Tremorine, first described by Everett (1956), produces a relatively long-lasting fine tremor, rigidity, asthenia, and hypothermia, in addition to a number of autonomic signs of which the parasympathetic, e.g., salivation, are more prominent. These effects are obtained to some extent in mice, rats, guinea pigs, dogs, pigeons, and monkeys (Farquharson and Johnston, 1959; Friedman and Smith, 1962; Trautner and Gershon, 1959; Everett, 1956; Everett e t al., 1956a; Frommel, 1958). Reactions in rabbit (Farquharson and Johnston, 1959), cat (Baker et al., 1960), toad (Trautner and Gershon, 1959) and frog (Friedman, 1962) are distinctly different from the above, varying from rage in the cat to sedation in the frog. Tremorine tremors occur both a t rest and during movement and exhibit a periodicity not unlike that observed in Parkinsonism. I n mice, the tremor involves the head, neck, limbs, and tail. Tail tremor has been reported by Farquharson and Johnston (1959) and, by connecting the tail to a force-displacement transducer and polygraph, is utilized by one of us (A.H.F.) as a means of quantifying tremor in mice. The onset of tremor is dose-dependent, appearing after a latency of 5-10 minutes with moderate intraperitoneal doses of 10-20 mg/kg, and within about a minute after 200 m&g. It is this latency which suggested that the formation of an active metabolite was involved. One metabolite obtained by incubation with liver slices (Kocis and Welch, 1960) was later identified as 1-(2-oxopyrrolidino) -4-pyrrolidino-butyne-2, or oxotremorine, by Cho and co-workers (1961) (Fig. 1). The onset of action of oxotremorine is prompt, and its potency appears to be considerably greater than that of the parent substance. It should prove to be a useful tool once the difficult synthesis is perfected to yield quantities adequate for extended studies. The duration of tremorine tremor in mice is about 3 hours, while in the dog and monkey it may last 48 hours (Everett, 1956). I n the latter species, the approximation of clinical Parkinsonism is remarkable in that such features as masked facies, cogwheel rigidity, and a propulsive gait can be observed. The tremorine-tremor frequency in rat and cat, respectively, are 8-1OJsecond (Blockus and Everett, 1957) and 8-l2/second (Nash and Emerson, 1959). I n man, Parkinson tremor varies between 3 and 9/second, averaging about 5/second (Hoefer and Putnam, 1940; Pelnar, 1913; Moldaver and Fairman, 1956). The frequency of physiological tremor in man is considered to be approximately twice that observed in the Parkinson patient (Hammond et al., 1956; Marshall and Walsh, 1956; Halliday and Readfern, 1958; Lippold et al., 1958). However, Marshall (1959, 1961) has shown that physiological tremor varies with age and that the frequency above the age of 40 declines from about lO/second to 6/second, so that Parkinsonian tremor may represent an exaggeration of the physiological tremor of age.

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The site(s) of action of tremorine in the production of tremor and rigidity has not been clearly defined. A subcortical site was inferred initially from studies of its action in decerebrate mice, rats, and rabbits, and the decerebellate dog (Everett et al., 1956a). Bernhang et al. (1958) found that lesions in the caudate nucleus of the rat raised the threshhold for tremorine tremor but did not change the quality of the response. The loss of tremor activity below the level of spinal transection was considered to be supporting evidence for a central action (Everett et d.,195613). However, Nash and Emerson (1959), using the chronic spinal cat, found that tremorine tremor could be produced below such a lesion. Kaelber and Hamel (1960) obtained negative results in the cat in a similar experiment, and suggested that the findings of Nash and Emerson were due to mass reflex phenomena. Chalmers and Yim (1962) subsequently were able to demonstrate hindlimb tremorine tremor in the chronic spinal rat following the recovery of the crossed extensor reflex. Dorsal root section or the administration of procyclidine abolished this tremor. Similar tremor phenomena were elicited in chronic spinal rats by arecoline and physostigmine, but not by prostigmine. These findings were considered to be an indication of tremorine action within the spinal cord, subject to feedback control. Further study is required to allow adequate interpretation of these apparently contradictory observations. Oxotremorine tremor is believed to be the consequence of a direct action in the central nervous system because (1) intraventricular injection in the rabbit produces a pronounced tremor; (2) spinal cord transection in the rat limits tremor to regions above the lesion; and finally (3) ablation of the midbrain tegmentum in the rat prevents its tremorigenic effect (George et al., 1962). 1. The Tremorhe Test The tremorine method of screening for anti-Parkinson compounds is relatively simple and clear-cut (Everett, 1964). Mice are pretreated with the test drug. One hour later, tremorine, 20 mg/kg, is administered intraperitoneally or subcutaneously. This is a dose which has been found to be 100% effective in producing full effects of tremor and salivation in untreated mice. Comparisons are made with standard compounds such as atropine, scopolamine, or trihexyphenidyl. These standard agents block tremorine effects in doses of 5-10 m@g for a period of about 4 hours. The arbitrary pretreatment interval of 1 hour eliminates shortacting drugs. Once an effective compound is found, the minimal effective dose and the duration of action may easily be determined. Special attention must be given to side effects of these potential agents. Compounds of some interest may also be screened in more advanced laboratory animals such as the dog or the monkey. Dosea of 2.5 mg/kg subcutaneously

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ALEXANDER H. FRIEDMAN AND GUY M. EVERETT

will usually produce adequate tremorine effects in both species although tremor itself is not marked in the monkey. With larger doses, tremorine effects become more severe and, in the presence of short-acting drugs, animals must be protected by repeated doses of cholinergic-blocking agents such as atropine. Trautner and Gershon (1959) reported that post-mortem examination of the brains of animals receiving tremorine revealed that the blood vessels were. dilated and that hemorrhages and clots were visible in the hemispheres and throughout the brain, especially subdurally or within the brain stem. We have also noted similar hemorrhages, but it has not been determined whether centrally acting cholinergic-blocking agents protect animals against this particular effect of tremorine, although Baker et al. (1960) reported that atropine prevented such changes in the cat. Established anti-Parkinson agents possess a relatively high specificity in antagonizing not only the tremorigenic and parasympathetic activity of tremorine (Everett, 1956, 1964; Keranen et al., 1961; Farquharson and Johnston, 1959; Bijlsma, 1957; Frommel and Fleury, 1958), but also its analgetic action (G. Chen, 1958; Keranen e t al., 1961) which in potency approaches that of morphine, The hypothermia produced by tremorine is partly controlled by some antiParkinson drugs (Keranen et al., 1961 ; Farquharson and Johnston, 1959), but only caramiphen appears to be effective in reversing it completely (Farquharson and Johnston, 1959). One exception to the findings reported above is that tiglyl-pseudotropine, which is said to possess anti-Parkinson activity (Trautner and Noack, 1951; Trautner and Gershon, 1958) and only weak cholinergicblocking activity, does not clearly antagonize the actions of tremorine (Trautner and Gershon, 1959). Although such a finding may detract somewhat from the value of the tremorine test, i t is clear that it “is a sensitive, useful and convenient method for screening anti-Parkinson drugs” (Vernier, 1963). It has been demonstrated that the central and peripheral effects of tremorine can be separated by means of quaternary cholinergic-blocking agents such as methanetheline (Everett, 1956; Trautner and Gershon, 1959; Farquharson and Johnston, 1959). However, Farquharson and Johnston (1959) have shown that quaternary tropine derivatives with distinct peripheral effects are capable of antagonizing tremorine tremor when administered in large doses and when sufficient time has been allowed for them to act. The specificity of tremorine antagonism is of a high order. Adrenergic-blocking agents of the a-type, which are effective against nicotine tremor (Cahen, 1953), are without effect against tremorine tremor or its peripheral actions (Everett, 1964). Anticonvulsants such as diphenylhydantoin, trimethadione, and phenacimide, or local anesthetics such as procaine are also ineffective. d-Tubo-

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curarine and mephenesin control tremors only a t paralytic doses, while ether is effective a t doses which produce anesthesia and exert some paralytic effect a t the neuromuscular junction. Although mice tranquilized by pretreatment with reserpine are not protected against tremorine effects (Everett, 1964), the tremor and analgesia appear to be reduced (A.H.F.) Chlorpromazine exerts a moderate anti-tremorine effect (Everett, 1964) ; Keranen et al., 1961; Ahmed and Marshall, 1962). This may be due to its anti-acetylcholine action (Ryall, 1956; Kopera and Armitage, 1954). On the other hand, Ahmed and Marshall (1962), in studying a series of phenothiazine derivatives, found that their antitremor effect was apparently nonspecific and seemed rather to be related to a general sedative effect of these compounds. It has also been suggested that some phenothiazine derivatives may exert their anti-tremorine effect by “merely inhibiting the conversion into the active metabolite” (Leslie and Maxwell, 1964). 2. Recent Studies Pharmacological actions of tremorine which have not been reported elsewhere, and which may ultimately help to explain the mechanism of tremorine action, should be mentioned. Friedman and Campos (1960) have found that tremorine produces in rats a hyperglycemia which is marked, and maximal in 1 hour. The effect is eliminated by adrenalectomy, adrenal demedullation, or splanchiectomy, indicating that the hyperglycemia is a consequence of a centrally mediated release of catecholamines. It is difficult to determine if this finding is pertinent to Parkinson’s disease. Recently Gates and Hyman (1960) found tolbutamide of value in paralysis agitans, although England and Schwab (1961a) have found no therapeutic value in this hypoglycemic agent. Glucose tolerance curves of postencephalitic Parkinson patients, studied by Barbeau et al. (1961a) were found to be elevated in 17 of 30 cases, and hepatic function tests were abnormal in 15. They also were unable to find any therapeutic value when tolbutamide was used alone in patients with idiopathic and postencephalitic Parkinsonism; however, when it was used in combination with Artane or Kemadrin there was, after several weeks, improvement in secondary symptomatology but not in tremor. The improvement did not coincide with any apparent change in glucose metabolism. Also, no improvement was seen in Parkinsonians following the administration of insulin. Tremorine has been shown to produce significant changes in biogenic amines of the brain of several species [Friedman, 1963; Friedman et al., 1963 (vide i n f r a ) ] . During the course of these studies it was noted that animals treated with tremorine exhibited a type of spinal shock upon

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decapitation by guillotine (Friedman, 1964). Decapitation of untreated rats, to cite one of these series, produces convulsive activity, which is largely clonic in nature, after a latency of 2.6+ 0.5 (S.D.) seconds. Tremorine-treated rats, on the other hand, convulse only after 23.5 k 6.1 seconds, and the convulsions are somewhat uncoordinated and tonic. The increase in the latency period is reproducible and very highly significant ( P < 0.001). The time course of the progressive increase in latency after tremorine follows the pattern of development of the intensity of tremorine tremor and rigidity. The prolonged latency is not altered by adrenalectomy, but is partly blocked by atropine. It is difficult to interpret the significance of this distinctive finding, but it suggests that tremorine may be exerting an inhibitory action on the spinal cord, although Baker (1963) has reported that tremorine does not have a significant effect on polysynaptic reflexes. (A surprising observation in mice, which seems to support the findings of Chalmers and Yim (1962) in the chronic spinal rat, is that following decapitation of tremorine-treated animals, fine tremors continue in the extremities for 10-15 seconds.) When decapitation latencies of groups of tremorine-treated rats were compared with those of groups treated with Z-dopa (dihydroxyphenylalanine) (50 mg/kg/ day, 3 days), 5-HTP (5-hydroxytryptophan) (50 mg/kg/day, 3 days), SKF 385 (10 mg/kg/day, 3 days), physostigmine (1.0 mg/kg, 1 hour pretreatment), reserpine (1.0 mg/kg/day, 3 days), and chlorpromaaine (25 mg/kg/day, 3 days), i t was noted that only in the case of reserpine and chlorpromazine was the magnitude of decapitation latency as great as or greater than that with tremorine. Decapitation latencies of the remaining groups, with the exception of physostigmine-treated animals which were somewhat increased, were not different from controls. Tremorh e , chlorpromaaine, and reserpine have in common, but to varying degrees, the ability to produce hypothermia and to influence the action of biologically important amines in the central and autonomic nervous system.

D. CHOLINERGIC-BLOCKING ACTIVITY 1. Isolated

Tissue Procedures

The in vitro antagonism of acetylcholine in isolated tissue preparations has been considered to be a valuable tool for the preliminary screening of anti-Parkinson drugs in viewrof the relative effectiveness of cholinergic-blocking agents in the treatment of Parkinson’s disease. Indeed Ahmed and Marshall (1962), using the isolated guinea pig ileum, found that the anti-acetylcholine potency of anti-Parkinson drugs correlated not only with their clinical value, but also with their ability to

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antagonize tremorine tremor. The anti-acetylcholine activity was always of a competitive nature. The behavior of chlorpromazine in this system was of a noncompetitive type. Although Frommel (1958) found that all anti-Parkinson drugs tested on the guinea pig ileum had some antihistaminic, antipapaverinic, and anticholinergic activity, with the latter predominating, he did not find complete parallelism between anticholinergic potency and anti-Parkinson effect. Similarly, Farquharson and Johnston (1959) concluded from their studies on the mouse pupil and guinea pig ileum that anti-Parkinson drugs possess considerable peripheral anti-acetylcholine activity. However, they were able to dissociate this from antitremor activity in their study of a series of acetyl tropeine derivatives. Furthermore they could find no correlation between the antitremor and the cholinolytic, antihistaminic, and local anesthetic activity of this series. It is possible that the lack of correlation may be related to differences in the ability of these compounds to pass through the blood brain barrier. DeJonge et al. (1960) also indicate that parasympatholytics have a restricted peripheral action, while anti-Parkinson agents are global in their antagonistic action. This dichotomy of action was already pointed out in 1956 by Everett. 2. The Electroencephalogram (EEG)

The use of the EEG has been suggested as a possible screening tool for anti-Parkinson drugs. It has been amply demonstrated that cholinergic substances such as acetylcholine and diisopropyl fluorophosphate (DFP), administered by close arterial injection, activate the cortex via the ascending reticular formation (Himwich and Rinaldi, 1955-1956), and that cholinergic-blocking agents, such as atropine, prevent the asynchronous, fast-wave alerting reaction and produce slow-wave cortical activity (Funderburk and Case, 1951), as do many of the agents used in the therapy of Parkinson’s disease. Kaelber and Correll (1958), using cats, and Everett (1964), utilizing unanesthetized restrained rabbits, were able to demonstrate that tremorine produced a desynchronized alert cortex, which was antagonized by chlorpromazine in the former study and by atropine in the latter, suggesting to Everett that the EEG effect of tremorine is result of ita central cholinergic action. Earlier, Jenkner and Ward (1953) demonstrated that diethazine could prevent the Parkinson-like tremor induced in monkeys by stimulation of the midbrain reticular formation. However, the question as to whether alerting phenomena and hyperactivity of the reticular formation are related to the production of Parkinson tremor and are only of cholinergic nature is difficult to answer when examined critically (Killam, 1962). White and Daigneault (1959) have shown that atropine was capable of

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inhibiting EEG-alerting phenomena in rabbits whether due to the action of cholinergic drugs such as physostigmine or the action of adrenergic drugs such as epinephrine, d-amphetamine, and methamphetamine, or to lesions produced in the brain. White and Westerbeke (1961), comparing the action of the anti-Parkinson agent ethopropazine (Parsidol) with that of several phenothiazines employed in psychiatry but known to produce extrapyramidal reactions, found that, in the rabbit, all of them antagonized the EEG and behavioral effects of d-amphetamine, but only ethopropazine (10 mg/kg) completely prevented alerting EEG effects produced by physostigmine or DFP, supporting the thesis that anti-Parkinson activity is produced by a central anticholinergic effect. The failure to detect any depressant action of five different adrenergic-blocking agents on the reticular activating system also has been considered evidence favoring the concept of cholinergic transmission in this region (Exley et al., 1958). Giarman and Pepeu (1962) have found that depression of the rat brain was associated with an increase in the total level of acetylcholine. Atropine and scopolamine decreased the level while producing behavioral changes suggesting excitement. Harris (1961) has also shown that cholinergic compounds such as pilocarpine, 2-dimethylaminoethyl acetate (tertiary amine analog of acetylcholine) as well as physostigmine decrease the spontaneous activity of mice, while various cholinergic-blocking agents such as atropine, scopolamine, and trihexyphenidyl increase the spontaneous activity and antagonize the depressed activity produced by the cholinergic compounds mentioned. It would be of interest to determine whether established anti-Parkinson agents produce their effects by an alteration of brain acetylcholine levels. EEG data alone do not provide an index of underlying mechanism. This was clearly shown by Wikler (1951), who demonstrated the dissociation between alert behavior and EEG “sleep patterns” in the atropinized dog. 3. Sinistrotorsion

A method for the evaluation of central anticholinergic activity is based on Forssman’s (1922) observation that the centripetal intracarotid injection of sheep-hemolytic rabbit serum into the guinea pig produced a torsion movement of the head. Diamant (1954) demonstrated that sinistrotorsion could be produced by the injection of cholinesterase inhibitors and that the effect probably resided in the brain stem. Although the injection of DFP into the semicircular ducts did not produce sinistrotorsion, labyrinthectomy and cerebellectomy did not prevent it. DeJonge and Funcke ( 1962) explained sinistrotorsion after the intracarotid injection of physostigmine on the basis of stimulation of the vestibular nuclei by the asymmetric acetylcholine distribution produced by local-

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ized cholinesterase inhibition. It is already considered that the efficacy of the relatively effective anti-Parkinson drugs is based on their cholinergic-blocking action (DeMaar, 1956; Ahmed and Marshall, 1962). The sinistrotorsion method produces quantized data, and is considered to be a good screening tool for anti-Parkinson agents since it gives good corroborative evidence with effective drugs. Sinistrotorsion appears to be antagonized specifically by known anti-Parkinson drugs (when they are administered 30 minutes before the injection of physostigmine) such as atropine, scopolamine, trihexyphenidyl, diethazine, caramiphen, benztropine, orphenadrine, and diphenhydramine (the latter is a potent antihistaminic and possesses local anesthetic activity, as well). Exception to this specificity is the finding that pentobarbital is also effective in blocking this response, as are large doses of iproniazid. Quaternized anticholinergic compounds are not effective antagonists nor are meprobamate, reserpine, chlorpromazine, bulbocapnine, impramine, amphetamine, or caffeine. It would be interesting to test the effectiveness of this technique after chronic rather than acute administration of some of the psychotropic agents listed above, in view of their ability to antagonize both drug-induced and true Parkinsonism (vide infra).

E. MISCELLANEOUS METHODS 1. Aminotripheny lpropanol Nine of a series of twelve amino alcohols synthesized by Ahmed et al. (1958) produce a profound, sustained coarse tremor accompanied by continuous propulsive, retropulsive, or circling movements in the absence of any evidence of parasympathetic activity. The most potent of these compounds is aminotriphenylpropanol (1,1,3-tripheny1-3-aminopropan1-01). This compound resembles trihexyphenidyl (benzhexol) , Artane, a potent anti-Parkinson drug which is capable of antagonizing both tremorine and nicotine tremor. Neither trihexyphenidyl and other antiParkinson drugs, nor cholinergic-, ganglionic-, and neuromuscular-blocking agents, analgetics, anticonvulsants, sedatives, reserpine, 5-hydroxytryptamine, and bulbocapnine afford any protection against aminotriphenylpropanol tremors. However, chlorpromazine, mephenesin, and pentobarbital do protect against such tremors. Day and Yen (1962) support some of these findings, adding chlorprothixene to the list of protective agents, but find that ganglionic-blocking agents, as well as trimethadione and bulbocapnine, antagonize amino alcohol tremors. yAminobutyric acid (GABA) has also been reported t o block such tremors (B6skoviE e t ul., 1960). As Ahmed et al. (1958) state, amino alcohol tremor may afford insight into the mechanism of tremor production, al-

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though i t is not a suitable tool for testing anti-Parkinson drugs. However, because of the basic dissimilarity between tremorine tremor and amino alcohol tremor, the latter might be found useful as a negative check in tremorine screening for anti-Parkinson agents. 2. Diethyl Cystearnine Koch and Hagen (1957) reported that a strong intention tremor could be induced in mice by the administration of small doses of diethyl cysteamine. Stern et al. (1961) confirmed these findings in mice, and found that this type of tremor could be more easily provoked by placing the animal on a rotating rod. None of the 65 compounds used, representing a wide variety of pharmacological agents, was found to be effective in antagonizing this dynamic type of tremor, although several potentiated it. This apparently indicates an entirely different mechanism for this type of tremor than for the static tremor of tremorine, harmine, and aminotriphenylpropanol. Stern et al. (1961) suggest that the action of diethyl cysteamine is central, since drugs with important central action, such as morphine, bulbocapnine, LSD, dopamine, and semicarbazide, potentiate intention tremor. They further suggest that the site of action may be in the cerebellum, If this is found to be true, it might be interesting to determine whether cysteamine will antagonize tremorine tremor, in view of Henner’s (1962) hypothesis of cerebellostriatal antagonism.

3. Gre-1248 Gre-1248 (l-hydroxy-3-acetamino-5-dimethyl-pyrrolidone-2) has been found to produce hyperkinesia in mice, which is characterized by running paroxysms, postural hindlimb tremor, and retropulsion (Bonta and Greven, 1961, 1962). These effects are seen only when the compound is given intracerebrally (12.5 mg/kg) . It is inert when administered intravenously. Antagonism studies with known anti-Parkinson drugs demonstrated that only two of eight tested, viz., orphenadrine and diethazine, actively protected mice challenged with Gre-1248. Since no other drug types were investigated to determine whether this compound exhibited specific action, one must, a t least for the present, consider this compound as an academic curiosity, rather than as a useful tool for screening anti-Parkinson drugs. 4. Veratramine Veratramine is a tremorigenic agent which differs clearly from tremorine and harmine in its spectrum of pharmacological activity. I n small doses it produces a coarse tremor and a t higher, sublethal doses, convulsive excitation. Schoetensack and Hallmann (1961) have shown that in

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rodents the tremor is blocked by only a few centrally acting muscle relaxants such as mephenesin, zoxazolamine, 2-aminobenzthiazole, chlormezanone, carisoprodol, meprobamate, and 2- (y-methoxypropylaminomethyl)-1 :4-beneodioxane-HCl. The latter two are the only ones in this group which do not block the convulsive action of veratramine. The highly selective activity of this compound is to be noted since other drugs such as ganglionic-, adrenergic-, and cholinergic-blocking agents, hypnotics, and anticonvulsants are without effect on either tremor or convulsion even with doses approaching lethal levels. The loci of activity, as determined by transection studies, appear to be in the lower brain stem and cerebellum. Whereas tremorine and harmine intensify decerebrate rigidity in the rat, veratramine produces a transient blockade of extensor hypertonus. It is apparent that veratramine cannot be used in a positive sense in screening for anti-Parkinson drugs, but may possibly be employed to exclude experimental drugs from more extensive studies. VI. Drug-Induced Parkinsonism

An examination of Pelnar’s comprehensive study of tremor (1913) reveals that a great many chemical substances are capable of evoking this response in man. Included in his compendium of tremorigenic agents are alcohol, ether, absinthe, carbon disulfide, hydrogen sulfide, iodine, bromine, chloral hydrate, carbon monoxide, arsenic, mercury, lead, chromium, zinc, tin, cadmium, copper, thallium, manganese, nicotine, caffeine, theine, opium, morphine, strychnine, curare, quinine, atropine, hyocyamine, colchicine, aconitine, cicutin (i.e., an extract of the rhizome of Cicuta V ~ T O S U ,the “water hemlock”), veratrin, physostigmine, pilocarpine, camphor, copaiva, ergot alkaloids, hashish, and poison mushrooms. Tremor has been demonstrated for many more substances since the publication of this classic work, as a casual perusal of any standard textbook of pharmacology will attest. It should be clear that the tremor produced by these agents is not necessarily equatable with Parkinson tremor, nor is there any unified theory that explains the tremorigenic action of such a diverse group of chemical substances. Iatrogenic Parkinsonism assumed importance only after the introduction of rauwolfia alkaloids and the halogenated phenothiazine derivatives. The latter are potent central sympathetic-blocking agents (Carlsson, 1959) while the former are potent depleters of catechol and indole amines (Brodie et ul., 1959). Although a genetic basis for the acquisition of true Parkinsonism may be indicated in some cases (Myrianthopolus et uZ., 1962), the suggestion that there may be a hereditary susceptibility to Parkinsonism produced by ataraxic drugs has not yet been clearly established (Allan, 1937; Mjones, 1949). It is known, however (Freyhan, 1957; Ayd, 1961; Gold-

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man, 19611, that the occurrence of drug-induced extrapyramidal reactions is not dependent on the total amount of drug received. This is so probably because of individual variation and the fact that drugs are usually administered on a fixed dose basis rather than on a milligram per kilogram basis. In the susceptible individual, extrapyramidal reactions may occur after only a single dose of a potent drug. Intolerance to one drug is often paralleled by an equivalent reaction to any others capable of producing extrapyramidal reactions. These reactions also appear to be unrelated to the age of the patient. In general, dyskinesia occurs in the younger, akathisia in the middle-aged, and Parkinsonism in the older group of patients. There is considerable overlap among the groups as well as scatter, so that one can find, for example, extrapyramidal drug reactions of all types in children. A causal relationship between Parkinsonism and the therapeutic effcct of the psychotropic agents is widely disputed (Kline and Mettler, 1961 ; Cole and Clyde, 1961; Goldman, 1961). The fact that one can obtain beneficial psychotherapeutic effects without producing Parkinsonism suggests the absence of such a relationship. The appearance of extrapyramidal reactions is more likely an indication that the patient is receiving full therapeutic doses of the psychotropic agent (Delay et d , 1959; Deniker, 1960). Threshold changes in motor function can be utilized to detect the appearance of extrapyramidal reactions. Haase (1954, 1955, 1961) utilizes the finer motor movements of handwriting for that purpose. Control of drug-induced extrapyramidal reactions is frequently achieved by withdrawing the causative agent, or by the use of standard anti-Parkinson drugs (Kruse, 1960; Goldman, 1961) or antihistaminics such as diphenhydramine (P. L. McGeer et al., 1961 ; Waugh and Metts, 1960). Freyhan (1959) found that caffeine sodium benzoate, administered intravenously, relieved dyskinetic disturbances within 10 to 20 minutes. Usually the dose requirement for the relief of drug-induced extrapyramidal reactions is lower than for true Parkinsonism. I n some instances the anti-Parkinson drug may relieve extrapyramidal reactions for weeks or months after it has been withdrawn.

A. RAUWOLFIA ALKALOIDS The chemistry and pharmacology of rauwolfia alkaloids has been reviewed extensively by Bein (1956) ; more recently Hollister (1961b) examined specifically the effects of these agents which might complicate their psychotherapeutic usage. It is already well established that these agents produce extrapyramidal reactions including the classic Parkinson syndrome (Bente and Itil, 1955; Bleuler and Stoll, 1955; De, 1944-1945; Flach, 1955; Huchtemann and Pflugfelder, 1955; Hughes e t al., 1955;

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Kinross-Wright, 1955; Kline and Stanley, 1955; Kline et al., 1956; Sainz, 1955; Weber, 1954; Ray, 1952; Steck, 1954; Margolies, 1957) in patients, and essentially identical effects have been produced experimentally in the monkey and chimpanzee by Windle and co-workers (1956; Windle and Cammermeyer, 1957). Himwich and Rinaldi (1955-1956) attributed extrapyramidal effects to a stimulatory action exerted by reserpine on the mesodiencephalic-activating system. Haley and Dasgupta (1959) have shown that intracerebral as well as intravenous injection of reserpine can elicit tremor activity in mice. The incidence of extrapyramidal reactions following chronic administration of reserpine is significant. Freyhan (19571, for example, has found that about 17% of his patients are affected in this manner, half of these by the twentieth day of therapy. The occurrence in the female patient of akathisia and Parkinsonism but not of dyskinesia is twice as great as that in the male (Ayd, 1961) whereas in true Parkinsonism the sex ratio is reversed (Doshay, 1960). An explanation for this reversed sex ratio after drug therapy may be that the female patient on an equal dosage schedule probably is receiving a greater amount of drug, on a milligram per kilogram basis, than is the male patient. This reversal of ratio is also true for phenothiazine derivatives. Akathisia occurs more frequently after reserpine than after chlorpromazine, and patients complain of “inner unrest.” Another distinction between reserpine and chlorpromazine is that sialorrhea occurs more frequently with the former. Reserpine and other neuroleptics may ameliorate true Parkinsonism and other hyperkinetic states (Lehmann, 1954; Trelles, 1954; Cotzias et al., 1961) ; however, a case of postencephalitic Parkinsonism in which death followed the administration of reserpine has also been reported (Freyhan, 1957). Among the naturally occurring and semisynthetic rauwolfia alkaloids, rescinnamine, syrosingopine, and methoserpidine are somewhat less potent centrally than is reserpine (Domino, 1962), and the latter two are more selective in their peripheral action. Orlans e t al. (1960) have reported that syrosingopine selectively depletes peripheral amines in the rabbit and dog without affecting central amines or producing sedation. Deserpidine, which differs from reserpine only in the absence of a methoxy group in the A ring, is similar in its pharmacological properties to the parent compound and produces a significant number of extrapyramidal reactions (Editorial, 1962). Among the synthetic pharmacological analogs of reserpine, the benzoquinolizine, tetrabenazine, has gained considerable attention as a tranquilizer (Zbinden, 1962). This short-acting compound selectively depresses the levels of serotonin and norepinephrine in the brain of the rabbit with only a limited effect peripherally (Quinn et al., 1959). Although peripheral toxicity is somewhat less with tetrabenazine

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than with reserpine (Voelkel, 1958), the occurrence of extrapyramidal side effects suggests that its central activity may be similar (Burckard et al., 1962). Benzquinamide (RO-2647),a benzoquinolisine that depresses conditioned avoidance behavior in rat and monkey, has a negligible effect on central nervous system amines, and not unexpectedly is also devoid of manifestations of parasympathetic activity (Finger et al., 1961). I n clinical trial i t has received praise as an effective antiemetic (Shulkin et al., 1962).However, with regard to its effectiveness in the treatment of psychoneuroses and psychoses, further study seems warranted in view of the fact that the range of doses used by different clinical investigators varies by a factor of as much as 10 (Shulkin et al., 1963; M.E.Smith, 1962,1963;Feldman, 1962) and the incidence of side effects reported ranges from virtually nonexistent to an occurrence of dystonia, akathisia, and Parkinsonism in as many as 36% of the subjects.

B. PHENOTHIAZINE DERIVATIVES Unsubstituted phenothiazine has been used as a vermifuge in veterinary medicine (Chenoweth, 1958),as a urinary antiseptic in man (De Eds et al., 1939),and a s an insecticide (Sollmann, 1942).Interest in compounds of this type was dormant until 1945 when the introduction of promethazine, a sedative antihistaminic, led to the synthesis of a series of phenothiazine derivatives including chlorpromasine. The report of Delay et al. (1952)on the usefulness of chlorpromazine in the treatment of agitated psychotics was largely responsible for the subsequent widespread use of the phenothiazine tranquilizers. Hundreds of related compounds have been synthesized and studied since that time, and more than a score of these are available for therapeutic application. Extensive bibliographical material has accumulated over the past decade attesting to the usefulness of these compounds and indicating the vast complexity of the pharmacology of the phenothiazine derivatives. Recent reviews (Domino, 1962; Hollister, 1961a; Kurland, 1962; Cares and Buckman, 1963; Parkes, 1961) have condensed much of this information into comprehensive form, and the material to follow is largely derived from these sources. Chlorpromazine is known as Largactil in France and England. This appellation is not surprising in view of the many properties shared by it and other substituted phenothiasines. In his review, Domino (1962) describes the following centrally mediated actions of the substituted phenothiasines: (1) sedation; (2) blockade of conditioned avoidance behavior ; (3) antiemetic effects; (4) altered temperature regulation; ( 5 ) altered tone of the skeletal muscles; (6) antipruritic effects; (7) analgesia; (8) facilitated seizure discharge; and (9) endocrine changes. I n the autonomic nervous system these compounds produce: (a) choliner-

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gic blockade at nicotinic and muscarinic receptor sites; (b) adrenergic blockade of the a-type; ( c ) potentiation of some adrenergic effects; and (d) antihistaminic effects and antiserotonin effects. In addition, they may potentiate the action of other drugs and produce local anesthesia.

Minor alteration in the molecular configuration of these compounds may profoundly influence not only their pharmacological action, but also the nature of their toxic effects, such as agranulocytosis, extrapyramidal reactions, jaundice, photosensitivity, and pigmentary retinopathy. Certain generalizations concerning the effect of structural change can be made with regard to their influence on incidence of such extrapyramidal reactions as Parkinsonism, athetosis, and chorea (Domino, 1962; Cares and Buckman, 1963; Psychophamcol. Serv. Center Bull., 1962;Ayd, 1960; Goldman, 1961). Substitutions on carbon 2 and the nitrogen appear to be most critical in determining a change in activity and potency. The incidence of Parkinsonism increases as central potency increases. Potency is enhanced by the substitution of a methoxy group or halogenation a t carbon 2. A trifluoromethyl substitution here engenders even greater activity. Increased potency is also found with acetyl and thio substitutions. However, in the case of the latter, as exemplified by thioridazine, the incidence of extrapyramidal reactions is decreased. The substitution in position 10 of a piperazine side chain for an aliphatic dimethylamino grouping also increases central activity and the tendency to produce extrapyramidal reactions. A two-carbon aliphatic side chain favors antihistaminic activity, while the addition of a third carbon increases adrenergic-blocking activity as well as general potency. The three-carbon internitrogen distance also appears to favor the antiemetic activity of substituted phenothiazines. An ethyl diethylamino substitution in position 10 converts the tranquilizing action of the phenothiasines into one of central stimulation. These derivatives, vis., diethazine and ethopropazine, have been shown to be useful in treating both true and drug-induced Parkinsonism. They are in addition antihistaminic and ganglioplegic agents. Imipramine and amitriptyline, iminodibenzyl compounds which are classified as antidepressants rather than neuroleptics, are structural analogs of the phenothiazines, and like them are capable of producing extrapyramidal reactions (Burt et al., 1962;Lambert et al., 1962;Druck-

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man et al., 1962). However, Mandell e t al. (1961) have utilized imipramine with considerable success in the treatment of Parkinson’s disease, especially in patients whose symptomatology is chiefly one of depression and poverty of movement rather than tremor. The effect is global, involving both the psychology and physiology of the patient, suggesting that motivation and ability to move are related phenomena. Extrapyramidal reactions are also produced by chlorprothixene, a thioxanthene analog of chlorpromaeine (Reznikoff, 1961; Barron et al., 1961). It differs from chlorpromazine in its psychotherapeutic spectrum in that it is effective in depressed as well as agitated psychoses (Kruse, 1959).

C. BUTYROPHENONES Most recent among the compounds which may be categorized as inducers of extrapyramidal reactions are the butyrophenones. These compounds (Table 111), synthesized by Janssen and co-workers (1959), are structurally unlike any of the compounds which currently enjoy popularity in psychiatry (although it is interesting to note that they bear some structural resemblance to one of the most potent tremorigenic agents known, i.e., tremorine). They have already gained considerable acceptance in Europe for the treatment of various neuroses and psychoses (Collard, 1961; Boissier et al., 1960; DeHaene, 1960; Delay e t al., 1960a, b,c; Chantraine et al., 1960; Gerle, 1960; Kristjansen, 1960; von Oles, 1960; Divry et al., 1958, 1959a,b; Schmitt and Schmitt, 1962; Brandrup and Kristjansen, 1961; Frommel e t al., 1960; Paquay et al., 1960) and are being used in the United States as well (Nodine e t al., 1962; Denber et al., 1959, 1962; Rajotte et al., 1962; Kabat, 1961). Among these, haloperidol ( R 1625) has been investigated most extensively. Divry et al. (1960a,b) report that this nonhypnotic drug is a valuable neuroleptic agent useful in all forms of psychomotor agitation including that produced in psychiatric practice by LSD, Metrazol, and electro-shock therapy. Inhibitory effects are obtained within 10 minutes after the oral administration of 5 mg and these effects last on an average of from 3-5 hours in 95% of the patients receiving this drug. The EEG pattern in man after such a dose is one of synchronization (Meurice, 1960). Extrapyramids1 reactions are frequently observed with all of the butyrophenones studied and a pseudo-Parkinsonian state may occur in which rigor, tremor, akinesia, drooling, as well as typical facies appear. Incidence as high as 80% has been reported (von Oles, 1960). Nodine e t al. (1962) found that the median toxic dose (TD 50) in ambulatory patients for compounds R 1625, R 1647, R 1892, and R 2167 (cf. Table I11 for structure) was 4,33,42, and 22 mg per day, respectively, analogous to the relative level of toxicity obtained in laboratory animals. At these dose levelti only mild Parkinson

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TABLE I11 BUTYROPHENONE DERIVATIVES Compound No.

Structural formula

Compound

Haloperidol

R 2498

Triperidol

Methylperidide

R 2028(Janssen,

0

A

R 2167 (Nodine et a2.,1962)'

a There appears to be

Haloanisone OCH,

a discrepancy in nomenclature

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ALEXANDER H. FRIEDMAN AND GUY M. EVERETT

reactions were observed. Withdrawal of these drugs causes a slow regression of these effects which has been found to persist for as long as a month after the medication was discontinued. Control of extrapyramidal effects has been achieved by anti-Parkinson drugs as well as by thioxanthine phenothiazines, imipramine, antihistaminics, mephenesin, and thiocholchicosine. Kabat (1961) has found that cerebellar tremor in the presence of haloperidol may be converted to a Parkinson-like tremor and that careful regulation of the dosage of butyrophenone, or medication with an anti-Parkinson drug (thiopropazate) held both the intention and resting tremor in abeyance. The effects on cerebellar tremor and ataxia persisted for as long as 6 months after the drug was discontinued. He suggests that haloperidol may act by virtue of its ability to depress facilitatory brain stem mechanisms in the diencephalon and basal ganglia. It has been demonstrated that lesions of the ventrolateral nucleus of the thalamus (Cooper and Poloukhine, 1959) may relieve not only cerebellar tremor but also the tremor and rigidity of Parkinsonism. This is attributed to the existence of common motor pathways for these conditions, as well as to a possible cerebellar-striatal antagonism (Kabat, 1959). With regard to the activity of other butyrophenone derivatives, triperidol (R 2498) is twice as potent as haloperidol in its psychotropic action. I n addition, it produces central excitatory effects not seen with haloperidol. Akathisia with tasikinesia occurs in 42% of patients as compared with 8% after haloperidol. Akinesia occurs more frequently with triperidol and a t lower daily doses, although the incidence of tremor is approximately the same as that seen with haloperidol (20%). Methylperidide (R 2963) has only half the neuroleptic potency of haloperidol. However, a t doses of 1&15 mg/day intramuscularly it induces akinetic hypertonia in 75% of the cases and an accompanying tremor in 2270. Parenteral administration is 3 to 6 times as effective as is oral administration. R 1647 (Divry et al., 1959b) has little therapeutic application and gives rather inconsistent results. R 2167 (Nodine et al., 1962) is to be noted because it is less likely to induce extrapyramidal reactions than haloperidol, although this compound produces a greater degree of hypotension. The hypotensive effect of the latter, originally thought not to be due to an adrenolytic action, has been shown by Schmitt and Schmitt (1962) to be due to both an adrenolytic and a small noradrenolytic action. Some interesting interactions of the butyrophenones with themselves and other compounds have been reported. Triperidol appears to increase the neuroleptic action of haloperidol as well as that of some of the phenothiazine derivatives, e.g., promethazine and prochlorperazine. Methylperidide antagonizes haloperidol-induced akathisia, although the reverse is not true; on the contrary, haloperidol reinforces that of methylperidide.

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Rajotte et al. (1962) found that haloperidol as well as chlorpromazine and thioperazine (all potent inducers of extrapyramidal reactions) inhibit mescaline-induced states, while diethazine (Diparcol), an important anti-Parkinson drug, tends t o aggravate the response t o mescaline. Whether this finding is of significance in the study of iatrogenic Parkinsonism and portends a useful screening procedure or is merely another fortuitous relationship is subject to conjecture. Certainly the striking ability of the butyrophenones to induce extrapyramidal reactions suggests that examination in depth might furnish important clues to the underlying mechanisms associated with extrapyramidal reactions. VII. Rational Pharmacotherapy

Modern experimental pharmacotherapy of Parkinsonism reflects a growing impression that a biochemical lesion may be an important factor in the etiology of the disease. I n a prophetic moment Pelnar (1913) expressed this idea when he said “. . . the disease is caused by a’poisonous substance which ceaselessly, for whole decades, acts on all systems of the organism, and is produced in the organism itself in either a definite organ or by a general pathologic metabolic process in that i t is an endogenous toxic process. . . .” Distinctive inclusion bodies (Lewy bodies) have been found in patients with Parkinsonism (Den Hartog Jager and Bethlem, 1960; Lipkin e t al., 1960; Bethlem and Den Hartog Jager, 1960) not only within the basal ganglia but also in the spinal cord and throughout the sympathetic ganglia. Occasionally Lewy bodies are extracellular. These findings are more apparent in idiopathic than in other variants of Parkinson’s disease, and since they have been found in other diseases their presence cannot be considered pathognomonic. A disturbance in ribonucleic acid (RNA) synthesis has also been implicated recently in Parkinsonism (Pakkenberg, 1962). The suggestion that liver function in Parkinson patients differs from the normal (Barbeau e t al., 1961a) has already been mentioned. The recent development of the biochemistry of biogenic amines has given impetus to studies which seek to uncover the underlying mechanisms of Parkinson’s disease. The distribution of various brain amines and some of the enzymes associated with their metabolism is presented in Table IV (from Sourkes, 1961). Norepinephrine is largely concentrated in the hypothalamus, mesencephalon, pons, and medulla (Sano et al., 1959). Dopa is not localized in any part of the brain but has its highest concentration in the nucleus ruber and choroid plexus. Serotonin (5-HT) is concentrated in brain stem structures, rhinencephalon, and neostriatum (Bogdanski e t al., 1957; Paasonen and Vogt, 1956; Costa and Aprison, 1958). Dopamine is localized in the putamen, caudate nucleus, pallidum,

TABLE I V CONCENTRATION OF SOME CONSTITUENTS OF

TEJC

BRAIN^

Monoamine oxidase

Decarboxylases Constituent Cerebral gray Corpus callosum Caudate nucleus Putamen Pallidum Internal capsule Substantia nigra Nucleus ruber Nucleus dentatus Thalamus Hypothalamus Midbrain Cerebellum Medulla oblongata

Dopa, m a d (mdgm) 0-30 10 20 30 20 30

5-HT, dogdVs 18

-

805

-

40 80 &70 -Y)-30 60

-

20 20

-

From Sourkes (1961). Sano et al. (1960). c Holtz and Westermann (1956). d Relative values. a Bogdanski et al. (1957). I Birkhauser (1941). 0

Dopa, oxCsd

100 163

-

100 304 258 84

Dopamine, manb (mmdgm)

Noradrenaline, man* (rnpglgm)

20-170 50 5740 8250 1010 380 380 1170 2U-30 300460 1120 0 170

Dogdue

Mand-1

040 0 40 70 20 40 70 230 0-20

87 100

-

40-90

100 173

1110 10 140

-

48

108 96 79

-

-

90 99 118

8:

PHARMACOLOGICAL ASPECTS OF PARKINSONISM

115

nucleus ruber, hypothalamus, and thalamus (Bertler and Rosengren, 1959; Carlsson e t al., 1958). Its unique distribution and the motor effects resulting from depletion by reserpine led Carlsson (1959) among others to consider the possibility that it had a specific function in the extrapyramidal tract. E. G. McGeer et a2. (1961) have shown that dopamine indeed is a powerful inhibitory substance; i t is 100 times more potent than GABA when tested on the stretch receptor neuron of the crayfish. Dopa, the immediate precursor of dopamine, will offset the effects of reserpine if given in the presence of a monoamine oxidase (MAO) inhibitor (Carlsson e t al., 1957; Holtz et al., 1957). A precursor of serotonin, 5-hydroxytryptophan (5-HTP), however, is ineffective. The importance of sympathetic amines in the maintenance of normal activity has been demonstrated by Everett et al. (1957), who restored the depressed activity of reserpinized mice with deoxyephedrine. Similar results were obtained in mice and monkeys (Everett and Toman, 1959) with a monoamine oxidase inhibitor and dopa. In the former, more depressing doses of chlorpromazine blocked the hyperactivity and central excitement. P. L. McGeer et al. (1961) postulated that the extrapyramidal side effects of reserpine or chlorpromazine may be due to interference with the physiological action of dopamine and that by giving precursors, one should be able to restore function. On the other hand, the most effective drugs in Parkinsonism, in general, are cholinergic-blocking agents or antihistamines, or they may combine both properties in a single molecule; benztropine methanesulfonate (Cogentin) is a good example of this combination. The high concentration of acetylcholine and histamine in the caudate nucleus (Hebb and Silver, 1956; Kopera and Laearini, 1953) suggests that this pair of biogenic amines may be components of a neutrotransmitter system which is balanced by catecholamines and 5-HT. In the case of drug-induced Parkinsonism, interference with the catecholamine-5-HT system would favor the acetylcholine-histamine system. Restoration of the imbalance might be achieved by the administration of anticholinergicJantihistaminic drugs or by the administration of compounds that could override the interference. I n practice, P. L. McGeer e t al. (1961) found that orally administered dl-dopa was only mildly beneficial, whereas diphenhydramine (Benadryl), an antihistamine possessing some anticholinergic activity (Reuse, 1948), controlled drug-induced extrapyramidal reactions in each of 11 cases in which it was used. A significant decrease in the urinary excretion of dopamine (Barbeau et al., 1961b) and 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of 5-HT (Barbeau and Jasmin, 1961), in Parkinson patients has been interpreted as an indication that Parkinsonism involves a metabolic dis-

116

ALEXANDER H. FRIEDMAN AND GUY M. EVERETT

order, although it is recognized that the brain content of these amines represents only 1% of their total amount in the body. The data of Greer and Williams (1963) show that the urinary excretion of homovanillic acid (HMV), the major terminal metabolite of dopamine, by Parkinson patients does not differ significantly from that of normal controls. However, their dopa loading study, which unfortunately is too limited for valid statistical interpretation, is interesting because the decreased excretion of HMV by the Parkinsonian suggests that his utilization of dopa is altered. Ehringer and Hornykiewicz (1960) and Bernheimer et aZ. (1961) found that the concentration of norepinephrine, dopamine, and 5-HT in the post-mortem brains of Parkinsonians was significantly decreased from the normal in regions where they are usually found in high concentration (although 5-HT levels in the reticular formation were unchanged). These biochemical changes are not unlike those seen in the animal brain after reserpinization (Holtzbauer and Vogt, 1956; Carlsson et al., 1958; Pletscher et aZ., 1956). This suggested that one might ameliorate Parkinsonism by restoring brain levels of these amines. Subsequently, Birkmayer and Hornykiewicz (1961) found that akinesia but not tremor was relieved for 3-24 hours by the intravenous administration of 1-dopa. Similar results were reported in preliminary studies with 5hydroxytryptophan (5-HTP), but further study by Birkmayer and Mentasi (1962) showed that neither 5-HTP, a serotonin procursor, nor dioxyphenylserine, a possible precursor of norepinephrine, had a therapeutic effect. They found that in the presence of a M A 0 inhibitor the therapeutic effect of Z-dopa was prolonged. Similar findings have also been reported by Barbeau (1962) and by Friedhoff et aZ. (1963), who found that transient relief of rigidity, but not tremor, could be obtained with Z-dopa (Fig, 2). Barbeau (1962) in addition demonstrated that the concomitant administration of a M A 0 inhibitor augmented the relief of rigidity but exaggerated the tremor. He has also shown that the disomer of dopa is ineffective and that the relief of rigidity by Z-dopa is antagonized by a-methyl dopa, an inhibitor of dopa-decarboxylase, and has postulated on the basis of these studies and earlier findings of decreased excretion of 5-HIAA in Parkinsonians that this disease may involve a relative deficiency in the enzyme, dopa-decarboxylase. Barbeau (1962) has formalized and modified McGeer’s hypothesis of biogenic amine equilibria by linking dopamine with acetylcholine; a disequilibrium in this system might result in production of rigidity and akinesia. The second system comprising 5-HT and histamine is considered to be concerned with tremor and akathisia. Many aspects of the hypothesis are still incomplete, especially with regard to the 5-HT-histamine system. As indicated earlier, the specific role of histamine in the central nervous

117

PHARMACOLOGICAL ASPECTS OF PARKINBONISM

system is unclear. It might be concerned with facilitating the transport of nutritional materials across cell membranes or i t might modulate the function of other biogenic amines in the brain. The importance of 5-HT in the maintenance of normal mental function was first suggested by Woolley and Shaw (1954) and by Gaddum (1954). This hypothesis has been fully documented in a recent book (Woolley, 1962). For arguments on whether the sedative action of reserpine is related to 5-HT or norepinephrine depletion the reader is referred to Brodie and Costa (1962), who favor the former and to Carlsson (1961), the protagonist for the

r "0'^

DOPA ALONE

200 mg

l4 11 10

PI

-

- zoo I" '240

RIQIDI1Y

--

-

-

8 .

n

b -

E 160

3 L

L o

-

. I

2

1'20

W . RECORDINO 4 -

L

YI

-

H3

40

latter. Although the role of 5-HT in the central nervous system as a neurohormone has not been convincingly determined (cf. discussion of Axelrod and Costa, 1962), a number of neurochemical and behavioral correlates are suggestive of this function, It has been demonstrated that an important determinant of the behavioral effect of 5-HT is its level in the brain (Brune and Himwich, 1961; Costa et al., 1960; Bogdanski et al., 1958; Carlsson et al., 1961). Moderate increases appear to be associated with tranquilization ; higher levels with tremor and convulsions. Domer and Feldberg (1960) recently demonstrated in cats that the intraventricular administration of 5-HT enhanced, while catecholamines depressed, drug-induced tremors; this observation correlates well with changes found to occur in the biogenic arnines of the rat brain after tremorine administration (vide infra) . An interesting observation of Bernheimer et al. (1961) is worth considering. They noted a lack of correlation between cellular degeneration

118

ALEXANDER H. FRIEDMAN AND GUY M. EVERETT

in serotonin-rich regions frequently damaged in Parkinsonism, such as the hypothalamus and substantia nigra, and the reduction of 5-HT found throughout all areas, other than the reticular formation, of the brains of these individuals. Since the glial cells are not numerically changed in Parkinsonism, one might consider that they are involved in the formation or storage of 5-HT, or some other function. I n this connection, Woolley’s group has demonstrated that serotonin increases the pulsating rhythmical movement of oligodendroglia (Benites et al., 1955), and suggests that one possible action of serotonin is to facilitate the passage of nutrients, oxygen, and waste products in regions of the brain where vascularisation is poor by changing pulsating rates of glial cells. Recently, experimental evidence supporting some of the clinical findings of Austrian and Canadian workers has been reported by Friedman (1963) and Friedman et al. (1963). They were able to demonstrate, using tremorine as a Parkinsonimimetic agent, that it produced an acute lowering of the concentration of catecholamines in the brain stem, cortex, and cerebellum of three species, the rat, mouse, and guinea pig, but failed to do so in a fourth species, the rabbit, which does not tremor after tremorine administration. The time course of these changes coincides in general with the development and intensity of tremor in each of the three species. In the rat, the changes in norepinephrine levels are followed by a progressive rise in brain stem 5-HT. Whether the change of the latter is of physiological significance or reflects metabolic depression due to the profound hypothermia produced by tremorine is a t present an open question. It is interesting that the motor behavior and biochemical changes observed under these experimental conditions reflect to some extent the clinical findings. From these studies it is apparent that rational pharmacotherapy depends on a more complete understanding of the biochemical and neurophysiological parameters involved. The development of tolerance to the more classic anti-Parkinson agents may be attributable to the fact that the therapeutic solution is only partial. A requirement for dopamine, for example, cannot be satisfied by the use of a cholinergic-blocking agent, although such a drug temporarily may limit the parasympathetic dominance, and restore the relative biogenic amine equilibrium between acetylcholine and catecholamine. VIII. Summary

Parkinsonism represents a complex of symptoms and etiologies, and insight into the underlying mechanisms requires the energies of many disciplines. Methods abound for the screening for satisfactory anti-Parkinson agents. Of these, the tremorine test is both simple and reliable, and therefore useful.

PHARMACOLOGICAL ASPECTS OF PARKINSONISM

119

A promising approach to rational pharmacotherapy of Parkinson’s disease has resulted from the discovery that a variety of widely used psychotropic drugs which produce extrapyramidal reactions also exert profound effects on the biogenic amines of the central nervous system. Until the focal defect of Parkinson’s disease is defined, multiple drug therapy of this disease will be required; it is necessary to treat not only inotor defects and autonomic reactions] but also the depression and anergia of the patient. Elucidation of such a primary cause may provide the possibility of the development of a single effective therapeutic agent. ACKNOWLEDGMENTS The authors wish to acknowledge the invaluable assistance given by Gertrud Friedman in the preparation of this manuscript. The work of A. H. Friedman was supported by Grant NB 03139-03,from the National Institutes of Health. REFERENCES Adey, W. R., Buchwald, N. A,, and Lindsley, D. F. (1960). Electroencephalog. Clin. Neurophysiol. 12, 21. Ahrned, A,, and Marshall, P. B. (1962).Brit. J. Pharmacol. 18, 247. Ahmed, A., Marshall, P. B., and Shepherd, D. M. (1958).J . Phamt. Pharmacol. 10, 672. Allan, W. (1937).Arch. Inlernal Med. SO, 424. A.M.A. Council on Drugs (1961).“New and Nonofficial Drugs, 1961,” p. 293. Lippincott, Philadelphia, Pennsylvania, Asai, K., and Hufschrnidt, H. J. (1958).Deut. Z.Neruenheilk. 178,289. Astlcy Cooper, H., and Gunn, J. A. (1931).Lancet 221, 901. Axelrod, J., and Costa, E. (1962).Ann. N . Y . Acad. Sci. 96,131. Ayd, F.J., Jr. (1960).Psychosomatics 1, 143. Ayd, F. J., Jr. (1961).J. A m . Med. Assoc. 175, 1054. Baker, W. W.(1963).Federation Proc. 22, 391. Raker, W. W.,Hosko, M. J., Rutt, W. J., and McGrath, J. R. (1960). Proc. SOC. Exptl. Biol. Med. 104, 214. Barbeau, A. (1962).Can. Med. Assoc. J. 87, 802. Barbeau, A,, and Jasmin, G. (1961).Rev. Can. Biol. 20, 837. Barbeau, A., Giguere, R., and Hardy, J. (1961a). Union M e d . Canada 90, 147. Barbeau, A., Murphy, G. F., and Sourkes, T. L. (1961b).Science 133, 1706. Rarraquer-Bordas, L. (1958).Encephale 47, 217. Barron, A,, Beckering, B., Rudy, L. H., and Smith, J. A. (1961). A m . J . Psychiat. 11% 347. Bein, H. J. (1956).Pliarmacol. Rev. 8, 435. Benda, C.E.,and Cobb, S. (1942).Medicine 21, 95. Benitez, H., Murray, M., and Woolley, D.W. (1955).Anat. Record 121, 446. Bente, D.G., and Itil, T. (1955).Med. Klin. Munich So, 1296. Rernhang, A. M.,Toman, J. E. P., and Everett, G. M. (1958). Abstr. Fall Meefing A m . SOC.Pharmacol. Exptl. Therap., Ann Arbor, Michigan, 1968, p. 4. Bernheimer, H., Birkmayer, W., and Hornykiewica, 0. (1961). Klin. Wochschr. 39, 1056. Bertler, A., and Rosengren, E.(1959).Acta Physiol. Scand. 47, 350.

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The Pharmacology and Biochemistry of Parasitic Helminths TAGE . MANSOUR Department of Pharmacology. Stanford University School of Medicine. Palo Alto. California

I . Introduction . . . . . . . . . . . . . . . I1. The Pharmacology of the Neuromuscular System . . . . . . A . General . . . . . . . . . . . . . . . B . Nerve Muscle Anatomy . . . . . . . . . . . C . Occurrence of Acetylcholine, Cholinesterase. and Cholineacetylnse . D . Occurrence and Synthesis of Serotonin . . . . . . . . E . The Effect of Neuromuscular Drugs . . . . . . . . F. The Effect of Serotonin and Lysergic Acid Derivatives . . . . I11. The Effect of Anthelmintic Agents on Neuromuscular System . . . A . The Effect on Nematodes . . . . . . . . . . . B . The Effect on Trematodes . . . . . . . . . . . C. The Effect on Cestodes . . . . . . . . . . . D . In Vivo Dislocation of Parasites by Drugs . . . . . . . IV . Carbohydrate Metabolism . . . . . . . . . . . A . General . . . . . . . . . . . . . . . B. Carbohydrate Utilization . . . . . . . . . . . C . Products of Carbohydrate Metabolism . . . . . . . . V . Control Mechanisms of Carbohydrate Metabolism . . . . . . A . General . . . . . . . . . . . . . . . B. Relationship between Energy Requirement and Carbohydrate Metabolism . . . . . . . . . . . . . . C . Regulatory Role of Phosphofructokinase . . . . . . . D . Control Mechanisms for Phosphofructokinase . . . . . . E . Regulatory Role of Phosphorylase . . . . . . . . . VI . Differences among Analogous Enzymes . . . . . . . . A . General . . . . . . . . . . . . . . . B . Kinetic Differences . . . . . . . . . . . . C . Immunological Differences . . . . . . . . . . . VII . Effect of Anthelmintic Agents on Carbohydrate Metabolism . . . A . General . . . . . . . . . . . . . . . B . The Antischistosomal Action of Antimonials . . . . . . C . The Biochemical Effects of Dithiasanine . . . . . . . D . The Antischistosomnl Action of Beneylic Diamines . . . . . E . The Antifilarial Action of Cyanine Dyes . . . . . . . VIII . Conclusion . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . .

129 130 130 131 131 133 134 135 137 137 139 140 141 142 142 143 144 146 146 145 148 151 154 155 155 156 156 158 158 158 159 160 161 162 162

.

1 Introduction

I n the past there has been a tendency to group all helminths. disregarding the phylogenetic differences among them . Since the work of the 129

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earlier classic biologists, who realized the great differences among members of this large group of animals, the helminths have been grouped into different phyla, according to their morphological resemblances. Among the helminths there are some which live as parasites on mammals during their sexually mature phase. These fall into two main phyla: the Nemathelminthes, e.g., A s m and ~ threadworm, and the Platyhelminthes which comprise two different classes: the trematodes (e.g., flukes and schistosomes) and the cestodes (tapeworms). In spite of the early recognition of the differences between members of different phyla, many biologists ignored this fact in their approach to the study of the effect of chemical agents on these organisms. Some of them used animals of a phylum other than those which include the parasitic helminths. Trendelenburg (1916) and Sollrnan (1919) used annelid material-mainly earthworms and leeches-to represent nematode parasites for their studies on the mode of action of anthelmintic drugs. The presence of a common physiological or biochemical principle in many forms of life has been responsible for such a unitary approach to the pharmacology of parasitic helminths. During the last two decades an increasing number of reports are now available in the literature demonstrating biochemical and physiological differences among various species of these organisms. The differences can be established not only by the presence or absence of a certain physiological or biochemical system but also by systems which carry on the same function in different parasites. As a result of these studies a rational approach to the problem of helminth chemotherapy is based on understanding the physiological and biochemical uniqueness of these organisms. Qualitative as well as quantitative differences between the host and the parasite are likely to be a basis for differential chemotherapy. It is the purpose here to review some of the recent studies concerning the carbohydrate metabolism and neuromuscular system of some parasitic helminths. The known effects of a relatively small number of chemical agents on these systems will also be discussed. I n any case this review is intended as an orientation to the subject and is not a coniplete discussion of all the latest work. II. The Pharmacology of the Neuromuscular System

A. GENERAL Survival of most parasitic helminths in their natural habitat is dependent on their ability to maintain themselves in situ in the face of peristaltic movement, in the case of intestinal parasites, or the movement of the blood or lymph, in case of some systemic parasites. Some parasites

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have specialized suckerlike organs to move within and attach themselves to the host. Most of these parasites can exhibit fast and well-coordinated rhythmical movement. These movements help the organisms to locate and maintain themselves in the host. Interest in the role played by the neuromuscular systems of these organisms in maintaining a successful parasitic life has provided the impetus for pharmacological studies similar to those carried out on isolated organ systems of vertebrates.

B. NERVE MUSCLEANATOMY According to Hyman (1951a) parasitic Platyhelminthes have a welldefined subcuticular musculature which consists of an outer circular, middle longitudinal, and inner diagonal layer. Mesenchymal musculature comprises dorsoventral, transverse, and longitudinal fibers. This muscular arrangement is probably responsible for the characteristic undulatory movement in these organisms (Mansour, 1949). This movement is characterized by a smooth, coordinated relaxation of the circular muscles at a point in the body, while a t the same time the antagonistic longitudinal and diagonal muscles contract. This results in shortening of the body. It is soon followed by extension of the body, due to the relaxation of longitudinal muscles and simultaneously a decrease in width brought about by the contraction of the circular muscles. The nervous system in trematodes mainly consists of two cerebral ganglia situated between the oral sucker and the pharynx. From these ganglia several nerves, usually three pairs, proceed anteriorly and three pairs-dorsal, lateral, and ventral-proceed posteriorly. I n cestodes the cerebral ganglia are located in the scolex from which a number of longitudinal chords run the full length of the body of the worm (Hyman, 1951a). Parasitic nematodes have underneath the cuticle a single layer of muscles consisting exclusively of longitudinal fibers, This muscle layer is divided by longitudinal chords into strips-two, four, or eight in number depending on the species (Hyman, 1951b). The presence of a single layer of longitudinal fibers is responsible for shortening or extending the body. The parasite can also be flicked dorsally and ventrally, sometimes in a loop. The main part of the nervous system consists of a ganglionic ring which encircles the pharynx. From this ganglion nerves run forward and six longitudinal nerves run to the posterior end of the body.

C. OCCURRENCEOF ACETYLCHOLINE, CHOLINESTERASE, AND CHOLINEACETYLASE The occurrence of acetylcholine in invertebrates was first demonstrated by Bacq (1935). Acetylcholine was reported to be present in extracts from

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the liver fluke (Fasciola hepatica) by Chance and Mansour (1953). The concentration of acetylcholine was determined to be as high as 0.19 to 1.7 pg/gm wet weight. This is nearly as high as the concentration of acetylcholine reported in the mammalian brain (McIlwain, 1959). The presence of cholineacetylase, the enzyme responsible for the synthesis of acetylcholine, was also demonstrated. Using assay conditions of Feldberg and Vogt (1948) fluke homogenates synthesized 4.3 to 12.7 pg/gm wet weight/ hour. Furthermore, homogenates of the same parasites can hydrolyze 48 pmole of acetyl-p-methylcholine chloride per gram wet weight per hour. No hydrolysis of benzoylcholine chloride was demonstrated. These results, according to the classification of Koelle and Gilman (1949) of cholinesterases, indicate the presence of a true cholinesterase in these organisms. The localization of this enzyme in the parasite has not been established. The presence of true cholinesterase as well as a weaker pseudocholinesterase was also reported in a related trematode parasite, Schist o s m a mansoni, by Bueding (1952). True cholinesterase was also demonstrated by the same author in two parasites: muscle of Ascaris lumbricoides and the whole worm of Litomosides carinii. The activity of acetylcholinesterase in Ascaris muscle was found to be considerably lower than that of the schistosomea. It is quite possible that the reason for this is the low content of nervous tissue in the isolated Ascuris muscle. Histochemical studies by Pepler (1958) demonstrated a true cholinesterase in the miracidium of Schistosoma mansoni. Localization of this enzyme appears to correspond anatomically most closely to the central nervous system of the miracidium. Artemov and Lure (1941) extracted a substance with the pharmacological properties of acetylcholine from Taenia

crassicollis and Dipylidium caninum. Acetylcholine has been reported by Bacq (1947) to be closely connected with motor activity among vertebrates as well as invertebrates. This was supported by Bulbring et al. (1949), who demonst,rated the presence of an acetylcholine system in the motile Trypanosoma rhodesiense and its absence in the nonmotile erythrocytic forms of malaria parasites. They suggested that the presence of acetylcholine might be related to the motility of parasites. Even in the mammalian heart muscle, where acetylcholine is supposed to have an inhibitory effect, Burn and Vane (1949) demonstrated that the activity of auricular muscles is closely related to its synthesis of acetylcholine, The results of experiments reported above on the flukes and on schistosomes, when added to the pharmacological effects of cholinergic drugs on these parasites (see Section 11, E) , suggest that acetylcholine and both its synthesizing and hydrolyzing enzymes play a functional role in these parasites.

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D. OCCURRENCE AND SYNTHESIS OF SEROTONIN The wide distribution of serotonin among invertebrate tissues has been reported by Erspamer (1954). Welsh and Moorhead (1960) demonstrated that serotonin is present in primitive nervous systems. The concentration of serotonin in the anterior part of the planarian worm body was found to be higher than that in the posterior part of the worm. The fact that the sense organs and ganglion cells are concentrated in the anterior part of the body of these worms might suggest the occurrence of serotonin in the planaria nervous organs. The presence of serotonin in the liver fluke Fasciola hepatica was demonstrated by Mansour et al. (1957). Acetone extracts from these organisms, when assayed on sensitized rat uterus preparations (Amin et al., 1954), contained 0.06-0.088 pg/gm wet weight (Table I ) . Furthermore, it was demonstrated that a system which catTABLE I THE PRESENCE AND SYNTHESIS OF SEROTONIN IN TEE LIVER FLUKEFasciola hepaticaa Serotonin synthesized per hour by fluke homogenates (pg/gm wet weight) ~

~~~~

Fresh intact flukes (pg/gm wet weight)

With pyridoxal phosphate

Without pyridoxal phosphate

0 . 060b 0. 077b 0. O8Sb 0.073'

182. 5b 142.V 146.Oc 137.Oe

80.00 38. Oc 75. Oc 71. Oc

~

Conditions for the synthesis of serotonin were those reported by Clark et a2. (1954). Bioassayed by the method of Amin et al. (1954). c Assayed by the colorimetric method of Udenfriend et al. (1955). a

alyzes the formation of serotonin from 5-hydroxytryptophan was present in these organisms, Incubation of homogenates of fresh flukes with substrate amounts of 5-hydroxytryptophan and pyridoxal phosphate for 1 hour resulted in the synthesis of 137-182 pg of serotonin/gm wet weight of flukes/hour (Table I). This evidence, when added to the fact that rhythmical movement of the liver fluke is increased by serotonin (Mansour, 1957), strongly suggests the presence of serotonin receptors which might influence motility of these organisms. The presence of similar receptors in closely related species is suggested since rhythmical movement of the following species can also be stimulated by serotonin: Schistosoma mansoni, Chlonorchis sinensis, and Taenia pisiformis (Mansour, 1958a). More investigations concerning the occurrence and synthesis of serotonin in these organisms are needed.

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MANSOUR

E. THEEFFECTOF NEUROMUSCULAR DRUGS Studies on the effectof drugs on the neuromuscular system of parasites have been carried out on isolated nerve muscle preparations. I n Ascaris, Baldwin and Moyle (1947, 1949) used nerve muscle preparations containing either the dorsal or the ventral nerve cord and a strip of muscle. Acetylcholine in low concentrations caused these preparations to Contract. Eserine by itself did not show an effect similar to that of acetylcholine. The effect of acetylcholine was not potentiated when tested on eserinized preparations. These results, when added to the finding that cholinesterase is present in these organisms (Bueding, 1952), might suggest that the enzyme in this tissue does not play the same role it plays in vertebrate neuromuscular systems. Nicotine a t low concentrations reproduced the effect of acetylcholine by causing the muscle to contract. It did not, however, in high concentrations, have the paralytic action which it displays in vertebrate tissues having nicotinic receptors. The effect of acetylcholine was antagonized by d-tubocurarine but not by atropine, nor was atropine alone active a t high concentrations. Thus it appears that the action of acetylcholine in this tissue is not muscarinic in nature. Adrenaline, atropine, ephedrine, histamine, and pilocarpine had no direct action on the Ascaris muscle. Similar studies were reported by Chance and Mansour (1953) on the liver fluke, Fasciola hepatica. Drugs which might affect the neuromuscular system were tested on two preparations from this parasite: the first preparation was the whole parasite and the second was prepared by cutting off the oral end of the organism. This provided a means of eliminating the cerebral ganglia which represent the brain in these organisms. The degangliated preparations had, after a brief period of time, rhythmical movement which was identical to that of the whole parasite preparation (Chance and Mansour, 1953). Furthermore, the response of the degangliated preparation to drugs was identical to that of the whole parasite preparation. This indicates that the coordination of rhythmical movement in these organisms is not central but peripheral. The choline esterscarbachol, acetylcholine, and methylcholine, in addition to eserinerelaxed these preparations with the end result either of complete paralysis or a marked reduction in the amplitude of contraction. Nicotine caused a rapid high contraction. The effect of the choline esters was reversible. Eserine sensitized the preparation to the action of acetylcholine. Atropine and curare were without effect on the fluke preparation but partially blocked the effect of acetylcholine and of carbachol. These results, when added to the fact that acetylcholine, true cholinesterase, and cholineacetylase are present in these organisms, strongly suggest the presence of

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cholinergic receptors which are responsible for relaxing these organisms. The above-mentioned results clearly indicate differences in the nature of the neuromuscular receptors of the parasite and the host. Even among parasitic helminths as a whole, differences between species with respect to response to neuromuscular drugs is well demonstrated in comparing the results obtained with a nematode with those obtained with a trematode. It is therefore not surprising that differences should also exist with regard to chemotherapeutic drugs effective against them.

F. THEEFFECT OF SEROTONIN AND LYSERGIC ACIDDERIVATIVES During an investigation on the mode of action of anthelmintic drugs on the liver fluke Fasciota hepatica by Chance and Mansour (1949), it was observed that rhythmical activity of both whole and degangliated preparations from this parasite was stimulated by sympathomimetic amines. The stimulatory potencies of these amines can be arranged in a descending order as follows : d-amphetamine, r-amphetamine, Z-amphetamine, P-phenylethylamine, phenylpropanolamine, ephedrine, and tyramine. Kephrine depressed the movement and epinephrine had no effect. The stimulatory potencies of these amines are surprisingly in line with their stimulant action on the mammalian central nervous system, as reported by Schulte et al. (1941). More recently it was reported by Mansour (1957) that rhythmical movement of the liver fluke was stimulated by 5-hydroxytryptamine (serotonin), lysergic acid diethylamide (LSD), and related indolealkylamines in concentrations much lower t.han those observed with amphetamine. The effect was peripheral and not mediated through the central ganglion. Bromolysergic acid diethylamide depressed rhythmical movement and antagonized the stimulant action of amphetamine, serotonin, and lysergic acid diethylamide. Furthermore, it was found necessary to increase the concentration of the stimulant whenever the concentration of the depressant drug was increased. These results suggest the presence of serotonin receptors in these organisms. This is strongly indicated when it was found that both serotonin and the system necessary for its synthesis are present in these oganisms (see Section I1,E). In a later investigation lysergic acid diethylamide derivatives and a number of indole compounds related to serotonin were tested on preparations from the liver fluke attached to a force transducer, and the concentrations which Stimulated 50% of these preparations (EDE0)were determined (Beernink et al., 1963). Among the derivatives tested lysergic acid diethylamide was the most potent compound, This is in accord with data reported on the psychomimetic action of this compound on man (Isbell et al., 1959) and the stimulant effect on the clam heart (A. M.

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TABLE I1 RELATIVEPOTENCIES OF LSD DERIVATI~ES A N D OTHERINDOLES RANKED IN DIFFERENTTESTSYSTEMS' Test system Rat uterusd

Clam heart'

Compounds

Plukesb

Man0

d-Lysergic acid diethylamide d-Lysergic acid pyrrolidide &Lysergic acid dimethylamide n-Methyld-lysergic acid diethylamide d-Lysergic acid monoethylamide d-Lysergic acid morpholide 1-Methyld-lysergic acid bu tanolamide Harmine Serotonin l-beneyl-2,5-dimethylserotonin 2-Bromo-d-lysergic acid diethylamide 5-Hydroxytryptophan Bufotenine &Lysergic acid diethylamide Lysergic acid

1.0 2.0 3.5 3.5

1.0 4.5 4.5 2.0

5.0 6.0 7.0

6.0 3.0

-

10

8.0 9.5 9.5 11.0

-

4

5-Hydroxyindoleacetic acid Rank order correlation with fluke data Significance level, P

12.0 13.0 14.0 15.5 15.5

-

1 -

6 3

-

-

7.0

7

8.0

9 2

-

0.764 o. 1

Data from Beernink et al. (1963). Ranked on the basis of concentrations which stimulated 50% of liver fluke preparations (Beernink et al., 1963). c Ranked on the basis of the dose of each drug which produced psychotomimetic effects in man equivalent to 1 pg LSD/kg (Isbell et al., 1959). d Ranked on the basis of dose of each drug which inhibits by one half a maximal contraction caused by serotonin (Cerletti and Doepfner, 1958). 0 Ranked on the basis of minimum molar concentrations of each drug which stimulated the heart of Venus marcenaria (Greenberg, 1960; A. M. Wright et al., 1962).

Wright e t al., 1962). A rank order correlation between the effects of these derivatives on the liver flukes and those reported on the clam heart, the rat uterus, and on man (Table 11) indicates that there is striking correlation between the relative effects of these derivatives on the flukes and on man. The correlation between the effect of LSD derivatives on the liver fluke and their effect on the clam heart and the rat uterus is much lower. Since there is a high degree of correlation between the psychotomimetic effects

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of lysergic acid diethylamide on man and their action on the rhythmical movement of the liver fluke, it is possible that these compounds act on systems in the flukes which are similar to those in the central nervous system of man. Studies, therefore, on the biochemical effect of these compounds on the liver fluke deserve special attention. 111. The Effect of Anthelmintic Agents on Neuromuscular System

A. THEEFFECT ON NEMATODES One of the approaches to the problem of studying the mechanism of action of anthelmintic agents has been to test these compounds in vitro on the motility of parasitic helminths. I n much of this work, kymograph tracings have been used to record effect on movement. Rebello and Rico (1926) were the first to recommend the use of isolated preparations from Ascaris which gave a good kymographic tracing when suspended in an organ bath. This kymographic technique was not used for a long time until it was revived by Baldwin (1943) who used small, tied-off neuromuscular preparations from the anterior and the intermediate part of pig Ascaris with an intact cuticle. H e demonstrated that these preparations can be used for in vitro screening of anthelmintic compounds against nematodes. Owing to its short duration, this procedure shows only the effect of chemical agents on the neuromuscular system of these organisms. By using the kymographic procedure Baldwin showed that there exists a high degree of correlation between anthelmintic potency in a given drug and its effect in paralyzing Ascaris preparations. Santonin, a n anthelmintic compound which was used specifically against nematodes was found by Baldwin to paralyze exclusively the anterior preparations a t low concentrations (1:50,000) and to stimulate the intermediate preparations of Ascaris a t a concentration of 1:5000. In view of this observation it was suggested that the mechanism of action of santonin is due to a paralytic action on the anterior part of the worm where the central nervous control is located, while stimulating the posterior part of the worm. This hypothesis was confirmed on preparations made of whole ascarids by Goodwin (1958). Thus santonin deprives the worm of the coordinating impulses which come from the central nerve ring. The end result of this action is presumed to be that the animal is no longer able to maintain its normal position in the host and can readily be expelled by giving a purgative. This explanation might account for previous reports that santonin does not have a direct killing action on intact Ascaris even when used a t concentrations as high as 0.1% (Lamson and Brown, 1936). Another evidence that anthelmintic drugs might owe their effect to an action on the neuromuscular system of the parasite is seen in studies on

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the mode of action of piperazine on Ascaris. This agent was shown to bc a potent anthelmintic against many nematode parasites. Standen (1955a) reported that intact Ascaris, when incubated with piperazine, were paralyzed. The paralytic action of the drug was reversed when the worms were subsequently transferred to a piperaeine-free medium. Furthermore, Ascaris expelled by patients who have been treated with piperazine were paralyzed, but their muscular activity was recovered when they were subsequently incubated in a piperazine-free medium (H. W. Brown et al., 1956; Swartzwelder e t al., 1955). Norton and DeBeer (1957) investigated the mode of action of piperazine on whole eviscerated Ascuris preparations. They reported that acetylcholine produced contraction of this preparation. The effect of acetylcholine was blocked by piperazine as well as by d-tubocurarine. Contraction induced by electric stimulation of these preparations was not antagonized by either piperazine or by d-tubocurarine. These experiments indicate that piperazine causes paralysis of Ascaris by directly blocking the neuromuscular junction. The action of piperazine on Ascaris neuromuscular junction was reported by Broome (1962) to be different from that of d-tubocurarine. In concentrations which blocked a given amount of acetylcholine, piperazine caused an immediate and sustained relaxation, while d-tubocurarine had little effect on spontaneous activity or muscle tone. These differences suggest that the two neuromuscular blocking agents might act a t different sites in Ascaris.

(I)

The possibility that methyridin (2-P-methoxyethylpyridine) (I), an anthelmintic with high activity against nematodes, acts on the neuromuscular junction of Ascaris was reported by Broome (1962). Using exposed neuromuscular preparations of Ascaris he showed that methyridin as well as acetylcholine causes rapid paralysis of rhythmical movement. The effect of both compounds was reversed by d-tubocurarine as well as by piperazine. The suggestion was made that methyridin produces a depolarizing effect on nematode muscle closely resembling that of acetylcholine. A similar effect was demonstrated in mammalian tissues, though at an excessive dose of the drug. Selective effect of this compound seems therefore to be dependent on a differential sensitivity of nematode and mammalian tissues to its action. Further evidence that anthelmintic drugs might act on cholinergic

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receptors in nematodes is supported by the fact that many organophosphorus compounds which have a potent anticholinesterase action have been shown to have anthelmintic activity (Broome, 1962). More experiments are needed to examine the mechanism of action of these compounds.

B. THE EFFECTON TREMATODES Chance and Mansour (1949) used a kymographic procedure to record the rhythmical movement of the whole liver fluke Fasciola hepatica and to test the effect of different substances on the movement. Depending upon the nature and, in some cases, upon the concentration of drugs, they could distinguish between three different types of action affecting movement: stimulant, paralyzant, and lethal. The stimulant drugs included amphetamine sulfate. A drug was classified as paralyzant if its action could be reversed by replacement of the medium with a solution containing amphetamine sulfate. Lethal drugs were those whose effects could not be reversed. Anthelmintic drugs which stimulate rhythmical movement of these preparations a t low concentrations but were lethal in high concentrations were carbon tetrachloride, tetrachloroethylene, hexachloroethane, and p-naphthol. It is possible that the superiority of the halogenated hydrocarbons as chemotherapeutic agents against the liver fluke could be partly due to stimulation of the rhythmical movement, thus dislocating the parasite from its natural environment. The second category of drugs were those which paralyzed the liver fluke preparations reversibly. These included arecoline, oil of chenopodium (in low concentrations), and pelletierine. The third category was those which paralyzed these preparations irreversibly and included oil of chenopodium (in high concentrations), thymol, hexylresorcinol, p-naphthol, carbon tetrachloride, tetrachloroethylene, hexachloroethane, extractum filix mas, and gentian violet. This technique was used for studying the relationship between the chemical structure of thiazine derivatives and their toxic and paralyzant effects (Mackie and Raeburn, 1952a,b; Mackie et al., 1955). Using the technique of Chance and Mansour (1949), it was found that the trivalent antimonials tartar emetic and stibophen were inactive against Fasciola hepatica in saline medium (Mansour, 1951). Tartar emetic, however, was lethal in a medium composed of equal volume of Ringer’s solution and bovine serum. The fraction of serum found to be responsible for this action was dialyzable through cellophane against distilled water but not against saline. This indicates the possible participation of the environment of the parasite on the effect of anthelmintic drugs. The chemotherapeutic activity of tris (p-aminophenyl) carbonium

140

TAG E. MANSOUR

salts (TAC) (11) against Schistosoma mansoni was reported by Thompson et al. (1962). Recently evidence was reported by Douglas et al. (1962) NH, I

X-

(n) suggesting that the anthelmintic action of this compound might be related to a neuromuscular effect on Schistosoma mansoni. Administration of subcurative doses of TAC resulted in a localized paralysis of the acetabulum and oral sucker. This effect was correlated histochemically with a pronounced decrease of cholinesterase activity in the central ganglia. The paralysis induced by TAC was blocked by ganglionic-blocking agent mecamylamine. It was then suggested by these authors that inhibition of cholinesterase activity by TAC would raise the acetylcholine levels in the central ganglia with the end result of localized paralysis in the acetabulum and the oral sucker. This is supported by the fact that cholinergic drugs have been reported to paralyze or reduce the muscular activity of a related trematode Fusciola hepatica (Chance and Mansour, 1953). Such effects might impair the mechanism by which the worm attaches itself to the host site or might impair the food ingestion process through the oral sucker so that the parasite is unable to maintain itself in the host.

C. THEEFFECTON CESTODES Betham (1946) used a kymographic technique to study the effect of arecoline on Taenia hydatigena and Taenia pisiformis. The activity of late mature or gravid proglottids of these worms was recorded. Solutions of arecoline hydrobromide (0.001% or more) caused relaxation and paralysis of the proglottid. The effect was reversible when arecoline solution was replaced by a few changes of fresh Ringer’s solution, indicating that the drug did not kill these preparations. Duguid and Heathcote (1950a,b) developed a similar in vitro kymographic technique using suitable lengths of Moniezia expansa suspended in tyrode solution to test the effect of different drugs. Using this procedure it was possible to confirm the paralyzant effect of arecoline on another cestode. Thus it appears that arecoline hydrobromide exerts its anthelmintic action by causing the

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muscles of cestodes to relax. Presumably the parasite’s hold on the host’s intestine is lost. The purgative effect of this drug on the host eventually results in the removal of the detached worms from the host’s body. That the anthelmintic action of arecoline was not due to its purgative effect was established by demonstrating that when the drug was injected subcutaneously it caused purgation but not worm removal (Betham, 1946). The work discussed above on the use of the kymographic technique with nematode, trematode, and cestode preparations has illustrated the two main approaches toward experimental chemotherapy of parasitic helminths. The first approach is derived from the observations that many of the already recognized anthelmintic drugs have a physiological effect on neuromuscular preparations of these organisms. A better understanding of the similarities as well as the differences between the neuromuscular physiology and pharmacology of the parasite might be of great value in finding more selective drugs which will affect the movement of the parasite without acting on the host. The other approach is the use of these procedures to study the relationship between a given effect on the parasite and a known variation of the molecular structure of a given molecule. Because of the difficulty of culturing the parasitic helminths in vitro, the kymographic procedures are good tools for such studies.

D. In Vivo DISLOCATION OF PARASITES BY DRUGS The evidence available in the literature suggests that some of the already known anthelmintic agents may affect in vivo the parasite neuromuscular function in a way which is not yet understood, but which by itself could make it impossible for the parasite to maintain itself in its natural habitat. Such an action was demonstrated by Rogers (1944), who showed how tetrachloroethylene caused the nematode parasite Nippestrongylus murk to leave the mucus of the rat’s intestine, after which they were expelled, while they were still alive, by the gut content movement. A closely related phenomenon was observed by Standen (1953) in his extensive studies on the mode of action of schistosomicidal agents. He observed that following treatment of experimentally infected rodents there was a shift in the distribution of the parasites from the mesenteric vessels to the liver. This was explained as being due to interference by t,hese agents with the muscle tone of the parasites with the end result that they are unable to hold onto the walls of the blood vessels and are gradually swept back with the portal blood to the liver. The actual deat,h of the schistosomes, according to Standen’s histological studies (1953), occurs in the liver. The walls of the vessels in which the worms lie became fibrosed and thickened and finally the lumen of the vessels was occluded. The worm is eventually killed and its tissues invaded by leucocytes. This

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TAG E. MANSOUR

effect was shown to be followed by remigration to the mesenteric veins in case drug elimination was accomplished before worm degeneration had proceeded too far. The latter process may explain the mechanism of relapse during the treatment of schistosomiasis (Standen, 1953). Standen (195513) investigated the mechanism of action of 1:7-bis ( p -

dimethylaminophenoxy) heptane (111).This is one of the diphcnoxyalkanes which was reported to have a high antischistosomal activity in experimental rodent infection (Raison and Standen, 1955; Caldwell and Standen, 1956; Standen and Walls, 1956; Gorvin et al., 1957). The effect on the distribution of the worms in the host followed the same pattern common to other schistosomicidal agents, namely, a passive shift of the worms from the mesenteric veins to the liver followed by ensheathment in inflammatory tissue and subsequent phagocytosis. There was, however, one characteristic effect in that the shift of the worms to the liver resulting from the diphenoxyalkanes was never followed by remigration to the mesenteric veins. An explanation can be obtained from histological investigation on the morphological changes which occur in the parasites after treatment with this compound (Standen, 1955b). It was demonstrated that the wider portion of schistosomes could be completely penetrated by phagocytes while the narrower anterior and posterior portions of the worms were unaffected. This suggested that the cuticle in the middle of the worms may be affected in some way which stimulates the typical foreign body reaction in the liver and the subsequent investment of the worms with phagocytes a t the mid-portion area first. Because of the ensheathing nature of the inflamed tissue the worms were unable to move away and phagocytic investment eventually involved the whole body. These studies confirm Watson’s view (1952) that an effective schistosomacide need not kill but need only incapacitate the worms. IV. Carbohydrate Metabolism

A. GENERAL The carbohydrate metabolism of many parasitic helminths has been investigated during the last 15 years. General reviews have appeared on the subject by von Brand (1952, 1960), Bueding (1949a, 1962a), and Read and Simmons (1963). I n general, these organisms have a high rate

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of anaerobic carbohydrate metabolism. This coincides with the anaerobic or semianaerobic environment of most of these parasites. Even among some parasites which live in an oxygen-rich environment, dependence on anaerobic carbohydrate metabolism has been demonstrated. Bueding (1950) showed that schistosomes which live in the blood depend almost entirely for their survival and reproduction on the anaerobic utilization of glucose. The amount of glucose utilized in 1 hour was equivalent to 15-26% of their dry weight. Among the many species studied so far, glycogen has been shown to be the main endogenous carbohydrate. Starvation either in vitro or in vivo results in a marked depletion of the glycogen content of many parasitic helminths. Glucose, when available in the outside environment, is utilized a t a high rate. The oxidation of carbohydrate by most of these organisms is not complete. This type of uneconomical metabolism results in the formation of organic acids which are not utilized by the parasites and are therefore excreted to the outside media. Studies on the carbohydrate metabolism of parasitic helminths, aside from its importance for our basic understanding of the comparative biochemical processes in nature, are also valuable for the rational development of more effective chemotherapeutic agents. Biochemical reactions which are found to be essential for the survival of the parasite and not for the host might be good targets for attack by chemical agents. B. CARBOHYDRATE UTILIZATION Experiments to measure the rate of carbohydrate metabolism have been carried out on organisms in a saline medium which can maintain their survival for a much longer period of time than the intended observation period. Recent experiments by Mansour (1959a) on the liver fluke, Fasciola hepatica, have indicated that anaerobic incubation of the worms in a medium containing no glucose resulted in a marked decrease in the total carbohydrate content. The initial amount of glycogen, found to be 3.6% of their wet weight, was reduced to 2% after starvation for 6 hours in saline medium a t 37OC. Worms which have been starved, when incubated in a medium containing glucose for the same length of time, showed an increase in the total carbohydrate which reached the same level as that of the initial. Thus glycogen synthesis and glycogen breakdown occur in these organisms a t approximately the same rate. Glycogen seems to be the most common polysaccharide among all the adult forms of parasitic helminths (von Brand, 1960). The concentration of glycogen, however, varies from one species to another. It can be as high as 8.7% in Ascaris lumbricoides (von Brand, 1952) and as low as 0.8% in Litornosoides curinii (Bueding, 1949b).

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TAG E. MANSOUR

Recently, Fairbairn and Passey (1957) and Fairbairn (1958) identified a nonreducing disaccharide, trehalose, which they have shown to be widely distributed among helminths. While relatively little trehalose was found in the liver fluke, Fasciola hepatica, and in cestodes, its concentration in the nematodes is high. The physiological importance of this sugar has not yet been determined. Fairbairn, however, suggested that it may share certain aspects of energy metabolism with glucose or substitute successfully for it. Many parasitic helminths live in environmcnts which contain glucose. Von Brand (1952) lists a variety of organisms which can readily utilize glucose. Starvation does not seem to alter the rate of glucose utilization by these parasites. This was indicated by the fact that in starved liver flukes the rate of glucose utilization was 23-25 pmoles/gm wet weight/ hour while in unstarved flukes it was 18-20 pmoles/gm wet weight/hour (Mansour, 1959a). Thcrcfore, the carbohydrate level in these organisms does not interfere with the rate of glucose uptake. Evidence that absorption of glucose in vitro by trematodes can occur entirely via the body surface was reported by Mansour (1959a). It was shown that the glucose uptake as well as the metabolic products of liver flukes, which had their oral openings closed by ligaturing between tlie anterior and posterior suckers, were identical to those of control flukes.

C. PRODUCTS OF CARBOHYDRATE METABOLISM Although there is a similarity between the carbohydrates utilized by these parasites and their hosts, the products of such metabolism are considerably different. A variety of metabolic products are formed by hclminths. Although lactic acid is produced by many species i t is not always the exclusive or the major metabolic product of carbohydrate metabolism. In Schistosoma mansoni, Bueding (1950) demonstratcd that over 80% of the metabolized glucose is converted to lactic acid. I n Dracunculz~s insignis lactic acid accounted for a t least 62% of the glucose utilized. Volatile fatty acids, higher fatty acids, succinic acid, and acetylmethylcarbinol have been identified as products of carbohydrate metabolism of many parasitic helminths. Metabolic balance experiments on the liver fluke Fasciola hepalica (Mansour, 1959a) have shown that the production of propionic and acetic acids accounted for almost all the carbohydrate utilized anaerobically. Only 4-8% of the metabolized carbohydrate was converted to lactic acid. The fluke also excretes an ether-extractable material which is, as yet, not identified, as well as large quantities of carbon dioxide. Metabolic carbon balance in this organism revealed that in tlie absence of glucose more products are formcd than can bc accountcd for by the utilization of carbohydrate. Therefore, in the absence of glucose

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from the medium the flukes must metabolize, in addition to glycogen, an undetermined endogenous metabolite. V. Control Mechanisms of Carbohydrate Metabolism

A. GENERAL Much information is now available concerning the fate of glycogen and glucose in parsitic helminths. It is self-evident that the chemical reactions in living cells do not proceed a t random rates. Now it is becoming essential not only to describe the different steps in a given pathway but also to analyze in detail the mechanisms that control them. I n 1956, C. F. Cori pointed out that “Biochemistry must inevitably move in this direction because it must ultimately deal with integrated enzymatic activity a t a cellular level of organization rather than with individual enzymes.” Studies over the past 10 years have demonstrated regulatory mechanisms that involve both enzyme formation and enzyme activity in bacteria as well as metazoan organisms. Genetic control over enzyme synthesis has been demonstrated in many single cell organisms. This is an important regulatory mechanism devised to meet changes in composition of the external environment. In metazoan organisms, however, where the external environment of the cell is somewhat more constant and the possibility of regulation by adjustment of concentration of enzymes is limited, the regulatory mechanisms lie mainly in the enzymes, their substrates, and cofactors. Regulation of metabolism in a parasite has to be adapted to the metabolic activity of the host tissue in order for the parasite to maintain a successful parasitic life. Studies on regulatory mechanisms in parasites have, therefore, been concerned not only with the parasite alone, but also with differences which exist between the parasite and the host cells. B. RELATIONSHIPBETWEEN ENERGY REQUIREMENT AND CARBOHYDRATE METABOLISM The problem of the relationship between the chemical reactions which provide the necessary energy for mechanical work of muscle has been the subject of many investigations in mammalian tissues. The fact that lactic acid fermentation can provide the energy necessary for work was reported in the classical work of Meyerhof (1920,1921). The reactions involved in lactic acid formation from glucose or from glycogen have been elucidated through his work and that of many others. Figure 1 represents the over-all Embden-Meyerhof scheme for glycolysis as it is understood today. I n parasitic helminths, in which the metabolism is associated with incomplete substrate oxidation, the question arises whether the chemical

146

TAG E. MANSOUR Glycogen + Pi

+ ATP

Glucose

Glucose 6-phosphate

t I - )1 1\1 1

(Phosphorylase)

: Glucose 1-phosphate

4

(Phosphoglucose tsomerase)

Fructose 6-phosphate

t

(Phosphogl~comutase)

+ ATP

(Phosphofructokinase)

Fructose 1,6-diphosphate (Aldolase)

Glyceraldehyde 3 -phosphate + DPN + Pi (Triose phosphate isomerase) (Glyceraldehyde 3phosphate dehydrogenase) DPNH 1,3-Diphosphoglyceric acid + ADP

Dihydr oxyacetone phosphate

-

(Phosphoglyceric phosphokinase)

i,,P 3-Phosphoglyceric acid (Phosphoglyceromutase)

2-Phosphoglycerlc acid (Enolaee)

t

Phosphoenolpyruvic acid + ADP (Phosphopyruvic kinaae) Pyruvic

1

A ‘T,

+ DPNH

(Lactic dehydrogenase) Lactic acid

FIG.1. Reactions involved in anaerobic glycosis of glucose and glycogen (EmbdenMeyerhof scheme).

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processes involved in providing the parasite with the necessary energy to meet extra work are the same as those in higher organisms. An answer to this problem might be obtained from studies reported by Mansour (1959b) on the liver fluke Fasciola hepatica. I n these experiments liver flukes were incubated anaerobically with serotonin in concentrations which increased their motility, and their metabolic balance was compared with that of control parasites. Stimulation of rhythmical movement in these parasites resulted in an increased glucose uptake or an increase in endogenous glycogen breakdown-when glucose was omitted from the media-and a two to tenfold increase in lactic acid production. The production of volatile fatty acids, which formed 91-96% of the metabolic products, was not significantly changed. Thus, stimulation of muscular activity by serotonin is accompanied by an increase in glycogenolysis and glycolysis. It appears then that the stimulation by serotonin of muscular activity produces a shift from fatty acids to lactic acid fermentation. These results indicated that in this parasite lactic acid fermentation can meet increased energy requirement more efficiently than can fatty acid fermentation. The question arises whether such an effect is due to stimulation by serotonin or could simply result from the increase in rhythmical movement. It was found that bromolysergic acid diethylamide could block both the effect of serotonin on rhythmical movement as well as the increase in lactic acid production, but could only partially antagonize the stimulatory action of serotonin on glucose uptake (Mansour, 1959b). These results imply that in addition to the influence of hyperactivity on the rate of lactic acid production, serotonin might have a direct effect on mechanisms concerned with the uptake or the utilization of glucose. This was supported by the fact that serotonin has an effect on a t least one enzyme involved in the utilization of glucose (see Section V, C). I n Ascaris lumbricoides, despite the demonstrations of systems catalyzing the formation of pyruvate via the Embden-Meyerhof scheme of glycolysis (Bueding and Yale, 1951), lactic acid is produced in insignificant amounts. Ascaris, however, produces large quantities of succinic acid under aerobic and anaerobic conditions (Bueding and Farrow, 1956; Bueding et al., 1959). The presence of succinate was demonstrated in the parasite as well as in the culture medium. I n their study on the biochemical effects of piperazine, Bueding e t al. (1959) demonstrated that during incubation of Ascaris with paralyzing concentrations of piperazine the formation of succinate both in the worm and in the medium is reduced. A good correlation between the paralyzing action of piperazine and its inhibition of succinate production was observed. Furthermore, the paralyzing effect of piperazine, as well as its inhibitory action on succinate

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production can be reversed by incubating the worms in a piperazine-free medium. Therefore, the ability of piperazine to cause paralysis in these worms parallels its ability to cause inhibition of succinate formation. The question of whether and in what manner the paralyzant effect of piperazine is related to its inhibitory action on succinate production was investigated by experiments on the rate of formation of succinate by Ascaris muscle strips. Saz and Vidrine (1959) reported that Ascaris muscle strips can incorporate lactate-2-C14 into succinate anaerobically followed by carbon dioxide fixation into pyruvate giving rise to malate. Conversion of malate to fumarate is catalyzed by a fumarase (Saz and Hubbard, 1957) followed by fumarate reduction to succinate. It was found by Bueding et al. (1959) that piperazine in concentrations which caused paralysis did not interfere with the incorporation of lactate-2-C1* into succinate by strips of Ascaris muscle. Thus the reduction in succinate production, though related to a reduction in muscular activity, is not caused by a direct effect of piperazine. It is possible, therefore, that succinate formation by Ascaris can meet the demand for high-energy requirement during normal muscular movement.

C. REGULATORY ROLEOF PHOSPHOFRUCTOKINASE The enzyme that catalyzes the formation of fructose diphosphate (FDP) from fructose 6-phosphate (F-6-P) and adenosine triphosphate (ATP) is one of the reactions involved in the Embden-Meyerhof glycolytic pathway for glycolysis and has been detected in many tissues. P-6-P

+ ATP

ME++ 4

FDP

+ ADP

The concentration of this enzyme has been found to be higher in tissues which convert carbohydrate to energy for work (Lardy, 1962). Thus skeletal muscle and heart muscle contain appreciable amounts of phosphofructokinase, while liver contains relatively little of this enzyme. The presence of the enzyme in parasitic helminths has been dcmonstrated in Schistosoma mansoni (Mansour and Bueding, 1954) and in Fasciola hepatica, Ascaris lumbricoides, and Taenia pisiformis (Mansour, 1962). Numcrous reports are now available in the literature indicating that phosphofructokinase plays a key role in the regulation of the metabolism of rapidly glycolyzing cells. C. F. Cori (1942) was the first to point out that phosphofructokinase is a rate-limiting enzyme in the processes involved in lactic acid formation. This was indicated by the findings of the Cori’s (G. T. Cori and Cori, 1936) that following incubation of frog muscle with epinephrine the endgenous concentration of the hexose monophosphate esters was increased to a greater extent than was lactic acid

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production. Furthermore, studies by C. F. Cori (1956) on the frog gastrocnemius muscle demonstrated that a marked accumulation of the hexose monophosphate esters was related to an increase in lactic acid formation during contraction following electric stimulation. Since in these experiments the sum of the hexose monophosphates and lactic acid formed can account for the amount of glycogen lost during contraction, it was assumed that other intermediates of glycolysis do not accumulate to an appreciable extent. The Cori’s experiments thus indicate that “. . . the reaction, glycogen + hexosemonophosphate, occurs more rapidly than the reaction, hexosemonophosphate + lactic acid and points to phosphofructokinase as the rate-limiting step for lactic acid formation” (C. F. Cori, 1956). The role that phosphofructokinase might play in explaining the Pasteur effect was suggested by Engel’hardt and Sakov (1943) who found that this enzyme is highly sensitive to oxidative agents. Based on the estimation of the steady state levels of glucose, glucose 6-phosphate (G-6-P), F-6-P, and FDP in the cell, several recent studies suggest that an incrcase in phosphofructokinase activity is responsible for the Pasteur effect. Lynen et al. (1959) reported that, in yeast cells, anoxia which causes an increase in rate of glucose phosphorylation also caused a reduction in the intracellular concentration of glucose and F-6-P and an increase in the intracellular levels of FDP. This suggests an increase in phosphofructokinase activity. Newsholme and Randle (1961) reported that, in the perfused anoxic heart, the concentration of FDP increased while the concentrations of G-6-P and F-6-P decreased. Furthermore, insulin increased the concentration of FDP without significantly altering the concentrations of G-6-P or F-6-P. Since there is little, if any, fructose l-6-diphosphatase in the rat heart, the enzyme that catalyzes the reverse of the phosphofructokinase reaction, (Newsholme and Randle, 1962) it is possible that both insulin or anoxia may exert, directly or indirectly, a stimulating action on phosphofructokinase in the heart. Evidence that phosphofructokinase is a rate-limiting reaction in Schistosoma mansoni was reported by Bueding and Mansour (1957). Using a cell-free extract from Schistosoma rnansoni they reported that addition of purified mammalian phosphofructokinase resulted in an increase in the rate of glycolysis when glucose was used as the substrate. The role played by phosphofructokinase in the regulation of glycolysis in the liver fluke, Fasciola hepatica, was studied by Mansour (1962). In these experiments the rate of lactic acid production from glucose, G-6P, F-6-P, and FDP was measured in homogenates from control flukes as well as from flukes preincubated with serotonin. As in the case of intact organisms (Mansour, 1962), the rate of glucose utilization and lactic acid

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formation from serotonin-treated homogenates was higher than with control homogenates. The increase in lactic acid production was also observed when G-6-P or F-6-P were used as the substrate. When FDP was the substrate, the production of lactic acid by control homogenates was markedly increased indicating that the concentration of this ester 60

50 h

.-C

62

.-0 a,

40

3

4-

a,

3

k 3c

\

a, -

r

3.

v

-0

'g 20 .-V

. I -

V

4 10

C

Glucose

G-6-P

F-6-P

FIG.2. Lactic acid production in homogenates from control and serotonh(5-HT) treated flukes in the presence of different substrates; glucose, G-6-P, F-6-P, FDP. Taken from the data of Mansour (1962).

limits the rate of glycolysis. The increase in lactic acid production in homogenates from flukes pretreated with serotonin was much less when FDP was the substrate (Fig. 2). These experiments indicate the following: ( a ) that phosphofructokinase is a rate-limiting enzyme since, when the product of this enzyme (FDP) was used, glycolysis proceeded a t a much higher rate than when F-6-P was the substrate; and ( b ) that serotonin

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increased the activity of this enzyme, since the stimulatory effect of serotonin was much reduced when F D P was used as the substrate. Further evidence that serotonin, in concentrations which increase glycolysis, also causes an increase in the activity of phosphofructokinase in the intact organism was gained by measurement of the levels of G-6-P, F-6-P, and F D P following incubation with serotonin (Mansour, 1962).

8-

6-

4-

2-

0-

d 0I

FDP FIG.3. Effect of serotonin ( 5 H T ) on the concentration of hexose phosphates in

;-6-P

the liver flukes. Taken from the data of Mansour (1962).

These results showed that under control conditions the concentrations of hexose monophosphate (G-6-P and F-6-P) were much higher than those of FDP. When glycolysis was stimulated by serotonin the levels of G-6-P and F-6-P fell while FDP levels rose indicating an increase in phosphofructokinase activity (Fig. 3).

D. CONTROL MECHANISMS FOR PHOSPHOFRUCTOKINASE One mechanism which might be responsible for controlling the activity of phosphofructokinase in the cell is that the enzyme might exist in an active and an inactive form. This would be similar to what had been reported before in the case of muscle phosphorylase (C. F. Cori and Cori,

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1945). This is suggested from studies on the stimulatory action of serotonin on phosphofructokinase from the liver fluke Fasciola hepatica. (Mansour and Mansour, 1962). It was demonstrated that direct addition of serotonin or cyclic 3‘,5‘-AMP to the fluke homogenate brought about an increase in phosphofructokinase activity. Since it was reported before that the formation of cyclic 3’,5’-AMP by broken cell preparations of the liver fluke was increased in the presence of serotonin (Mansour et al., 1960) the hypothesis was considered that the effect of serotonin is indirect and might be mediated via cyclic 3’,5’-AMP. This was confirmed by the finding that phosphofructokinase activation by serotonin is dependent on the presence of the same particulate fraction required for cyclic 3’,5’AMP synthesis (Mansour and Mansour, 1962). Activation by the cyclic nucleotide, on the other hand, was demonstrated in a soluble fraction in the presence of ATP and Mg++and in the absence of the particulate fraction. Phosphofructokinase activation in these experiments was not dependent on the presence of cyclic 3’,5’-AMP during the enzyme assay. Concentrated enzyme preparations, which were incubated with ATP, Mg”, and cyclic 3’,5’-AMP and assayed after severalfold dilution, were activated. Under these conditions, the concentration of cyclic 3’,5’-AMP in the assay mixtures did not have a direct effect on the enzyme during assay. Cat+ was also reported to activate phosphofructokinase (Mansour and Mansour, 1962). This effect was shown to be dependent on the prcsence of a heavy particulate fraction, ATP and Mg++.Thc similarity of this system to that reported by Krebs et al. (1959) for the activation of phosphorylasc-b-kinasc in rabbit skeletal muscle is striking. Both Ca++ and cyclic 3’,5’-AMP have been reported to activate rabbit musclc phosphorylase b-kinase and liver fluke phosphofructokinase. The nature of activation of the fluke phosphofructokinase by cyclic 3’,5’-AMP is still undetermined. The possibility exists that there might be a change in the molecular entity of the enzyme. Cyclic 3’,5’-AMP in low concentrations, under conditions similar to those used for the liver fluke, activated phosphofructokinase from two other parasitic flatworms, Schistosoma mansoni and Taenia pisiformis (Mansour and Mansour, 1962). On the other hand, a lesser activation by the cyclic nucleotide was observed on phosphofructokinase from muscle homogenates of Ascaris lumbricoides. It is possible that a mechanism for the control of phosphofructokinase, similar to that in the flukes, is present in other parasitic worms. Another mechanism which might control the activity of phosphofructokinase in the cell is a characteristic property of the kinetics of this enzyme. It was first discovered by Lardy and Parks (1956) that ATP

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in excess of the molar equivalent of Mg++present inhibited the rate of skeletal muscle phosphofructokinase. Similarly, phosphofructokinase from Schistosoma mansoni (Bueding and Mansour, 1957), Fasciola hepatica (Mansour and Mansour, 1962), brain (Passonneau and Lowry, 1962), and heart muscle (Mansour, 1963) were shown to be inhibited by ATP. It was possible to antagonize the ATP inhibition in Fasciola hepatica phosphofructokinase by cyclic 3’,5’-AMP (Mansour and Mansour, 1962) and, in the case of the mammalian enzyme, by cyclic 3’,5’-AMP, AMP, ADP, and Pi (Passonneau and Lowry, 1962; Mansour, 1963). The kinetics of phosphofructokinase, when inhibited by ATP or activated by cyclic 3’,5‘-AMP, were investigated on a highly purified enzyme preparation from the heart (Mansour, 1963). It was reported that the purified enzyme was strongly inhibited by ATP a t pH 6.9 and in the presence of low concentrations of F-6-P. Cyclic 3’,5’-AMP caused a marked increase in the enzyme activity only when the enzyme was inhibited by ATP. Kinetic data indicated that ATP is partially competitive with respect to cyclic 3‘,5’-AMP. The cyclic nucleotide caused a decrease in the concentration of F-6-P necessary for half-maximal activity of the inhibited enzyme. One of the characteristic features of activation by cyclic 3’,5’AMP is that it results primarily from a marked decrease of the Michaelis constant for F-6-P. When cyclic 3’,5’-AMP was tested in the presence of high concentrations of F-6-P no activation occurred. Similar kinetic data were reported for enzymes in bacteria which are subject to feedback inhibition (Umbarger, 1961; Changeaux, 1961; Gerhardt and Pardee, 1962). A model based on the assumption that the enzyme combines with one molecule of ATP a t the active site and one molecule or more of ATP at other sites on the enzyme which are designated an inhibitory site or sites might explain these kinetics. Activation of the ATP-inhibited enzyme by cyclic 3’,5’-AMP and related compounds can be explained on this multisite model by assuming that the activators compete with ATP for the extra sites and that the activator-enzyme complex is fully active with respect to F-6-P. This was found to be consistent with the finding that ATP inhibition was partially competitive with respect to cyclic 3’,5’-AMP (Mansour, 1963). These kinetic data strongly suggest that the intracellular levels of ATP and the activator nucleotides could control the activity of this enzyme. The intracellular concentration of ATP in the heart muscle (Hochrein and Doring, 1958), assuming uniform M . Under conditions M to 8 X distribution in the cell, is 7 X which might well exist in the cell (pH 6.9, low F-6-P) such ATP concentration is high enough to completely inhibit the enzyme. If this is the

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TAG E. MANSOUR

case, then one would expect that fluctuations in the levels of adenine nucleotides (5’-AMP, cyclic 3’,5’-AMP, ADP) and inorganic phosphate might result in an activation or inhibition of the enzyme in the cell.

E. REGULATORY ROLEOF PHOSPHORYLASE Since glycogen is the main source of energy for many parasitic helminths attention should be given to the enzyme that catalyzes the conversion of glycogen to glucose l-phospate (G-1-P) , namely, phosphorylase. A characteristic property of this eneyme in mammalian tissues is its capacity to undergo reversible inactivation through the action of two enzymes. The two forms of phosphorylase are designated as phosphorylase a for active form and phosphorylase b for the inactive form. Two enzymes are involved in catalyzing the interconversion of the two forms of phosphorylase. A specific kinase transfers phosphate from ATP to the serine residue in phosphorylase b. A specific phosphatase splits phosphate from phosphoserine groups on phosphorylase a (D. H. Brown and Cori, 1961; Rall and Sutherland, 1961; Krebs and Fischer, 1962). The importance of the phosphorylase control systcm for the regulation of glycogenolysis was discovered in the course of studies on the cellular effect of epinephrine on the cell. Sutherland and Cori (1951) and Sutherland (1951) established that the increased glycogen breakdown in rabbit liver slices and in rat hemidiaphragrns induced by epinephrine is associated with an increase in the amount of active phosphorylase. It was later discovered that in a cell-free extract epinephrine can displace the balance of the phosphorylase interconversion reaction in favor of active phosphorylase (Rall et al., 1956). The response to epinephrine was separated into two phases: first, the formation of cyclic 3’,5’-AMP from ATP catalyzed by adenyl cyclase ; second, the activation of phosphorylase kinase which will increase the formation of active phosphorylase ( Sutherland and Rall, 1960). Thus the effect of epinephrine is an indirect one and appears to be mediated through the production of cyclic 3’,5’-AMP. I n the liver fluke, Fasciola hepatica, a n increase in glycogenolysis following stimulation of muscular activity by serotonin (Mansour, 195913) suggested that in these organisms a system analogous to that found in mammalian tissues might exist. Experiments carried out on homogenates from these organisms indicated that phosphorylase is reversibly converted to active and inactive forms (Mansour e t al., 1960). Inactivation can be achieved by incubation of the homogenates for 10 minutes a t 3OOC. Reactivation of this enzyme was demonstrated when homogenates containing the inactive enzyme were incubated with ATP and Mg”. When the intact flukes were incubated with serotonin, an increase in glycogen

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breakdown and lactic acid production was associated with an increased level of active phosphorylase (Mansour et al., 1960). None of these effects can be induced by epinephrine or its congeners. Using conditions similar to those used by Sutherland and associates for the synthesis of cyclic 3’,5’-AMP in mammalian tissues, i t was observed by Mansour et al. (1960) that serotonin caused a striking increase in the formation of cyclic 3’,5’-AMP by particulate fractions of the liver fluke while neither epinephrine nor norepinephrine had any effect. The adenosine 3‘,5’-phosphate produced in the presence of the liver fluke particulate fractions, when isolated and purified, was found to be identical with that formed by mammalian tissue particles. Similar experiments were carried out with Schistosoma mansoni and Ascaris lumbricoides. Using schistosomes, in 1 out of 13 experiments some apparent effect was noted, but the response was not striking. I n the case of Ascaris Zumbricoides no increase in the formation of cyclic 3’,5’-AMP by serotonin or epinephrine was observed. Sutherland and Rall (1960) reported that the synthesis of cyclic 3’,5’-AMP can be demonstrated in a wide variety of tissues. However, there is a certain specificity regarding the hormone which promotes the synthesis of this nucleotide. For example, ACTH increased phosphorylase levels and cyclic 3’,5’-AMP in the adrenal cortex (Haynes, 1958) and glucagon had a similar effect on liver. Since the presence of serotonin in F . hepatica has been demonstrated (Mansour et al., 1957), it seems possible that serotonin plays a role in this organism similar to that of epinephrine in some tissues of higher organisms. Whether a similar role can be shown for serotonin in other parasitic helminths and invertebrates has to await further investigations. Such a role, however, can be speculated upon since serotonin rather than epinephrine has been implicated as the neurohumoral transmitter in some invertebrate heart preparations (Welsh, 1953). VI. Differences among Analogous Enzymes

A. GENERAL Enzymes constitute, by themselves, a controlling factor in the breakdown of substrates in the cell. It is a well-known fact that the rate of an enzymic reaction is dependent on its physical kinetic constants. Concentration of the substrate, pH, redox potential, and temperature are all important factors which influence enzyme activity. Changes in the activity of one enzyme might result in a change of the activity of other enzymes. Enzymes from different species, although they catalyze the same reac-

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tions, are not identical. Differences between analogous enzymes can be observed in their kinetic properties, their sensitivity to inhibitors, or their behavior to enzyme antibodies.

B. KINETICDIFFERENCES Studies on some of the glycolytic enzymes in several parasitic helrninths revealed that the kinetic properties of these enzymes are different from those of analogous mammalian enzymes. Kinetic properties of lactic dehydrogenase of Schistosoma mansoni when compared with those of the same enzyme from the rabbit muscle (Mansour and Bueding, 1953) indicated that the two enzymes are not identical. The pH optima for the schistosome enzyme were significantly lower in either direction of the reaction. The dissociation constants and the optimal concentration for pyruvate of the worm enzyme were 6-12 times higher than that of the mammalian enzyme. Bueding and MacKinnon (1955a) reported that Schistosoma mansoni contains four distinct hexokinases, each one of which reacts specifically with glucose, fructose, mannose, or glucosaminr. This is in contrast to yeast hexokinase and brain hexokinase which catalyze the phosphorylation of the first three substrates. I n addition to differences in the substrate specificity, the glucokinase of the parasite differs from brain hexokinase by its higher dissociation constants for glucose, ATP, and Mg++.Furthermore, in constrast to fructokinase of mammalian liver and muscle, schistosome fructokinase is inhibited by glucose and does not catalyze the phosphorylation of l-sorbose. Differences between phenol oxidase from the liver fluke and phenol oxidase from the mouse melanoma were reported by Mansour (1958b). While the fluke enzyme catalyzed the oxidation of l-epinephrine a t a high rate and dihydroxyphenylalanine a t a low rate, the reverse situation was observed in the case of phenol oxidase from mouse melanoma. Serotonin inhibits both enzymes, but the inhibition was competitive in the case of the fluke enzyme and noncompetitive with the melanoma enzyme.

C. IMMUNOLOGICAL DIFFERENCES Enzymes catalyzing the same reactions in different cells may have identical substrate specificity and similar kinetic properties but have different immunological properties. This indicates that these enzymes are not identical. The first example of immunological differences among some of the glycolytic enzymes was reported by Krebs and Naj jar (1948). They demonstrated that rabbit, antisera to glyceraldehyde 3-phosphate dehydrogenase of yeast inhibited the activity of this enzyme while the same sera did not affect glyceraldehyde 3-phosphate dehydrogenase of rabbit muscle. Similar experiments were carried out by Mansour et al. (1954)

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in an attempt to distinguish between lactic dehydrogenase of schistosomes and the same enzyme from rabbit muscle. When rabbit muscle lactic dehydrogenase was used as an antigen in roosters, the antiserum obtained, although inhibitory t o the enzymic activity of the rabbit enzyme, was less inhibitory to the activity of rat muscle lactic dehydrogenase and completely without effect on the same enzyme from Schistosoma mansoni or from Schistosoma japonicum. Neither diphosphopyridine nucleotide (DPN) nor pyruvate protected lactic dehydrogenase from inhibition by the antibody, indicating that the antibody does not necessarily exert its inhibitory effect by combining with the same site on the enzyme as the substrate and the coenzyme. The specificity of the enzyme-antibody reaction was demonstrated by the observation that immune serum against rabbit lactic dehydrogenase did not affect the activities of three other glycolytic enzymes of rabbit muscle-phosphohexose isomerase, aldolase, and phosphoglyceraldehyde dehydrogenase. Antiserum against lactic dehydrogenase of Schistosoma mansoni inhibited the activity of this enzyme as well as lactic dehydrogenase from Schistosoma japonicum and from Schistosomu hematobium (Henion et al., 1955). The same immune serum had no effect on the activity of lactic dehydrogenase of rabbit muscle or on the activity of another glycolytic enzyme of Schistosoma mansoni, phosphohexose isomerase. D P N H protected the schistosome enzyme from the inhibitory effect of the immune serum, suggesting that a t least one point of attachment of the antibody on the enzyme is located a t the site of the active center where the combination with DPNH occurs. A similar species specificity was observed by Bueding and MacKinnon (1955b) comparing the phosphoglucose isomerases of S. mansoni and rabbit muscle. Aside from the species specificity among enzymes, differences among analogous enzymes from the same animal but from different tissues have been reported. Kaplan et al. (1960) demonstrated kinetic as well as immunological differences among lactic dehydrogenases from three human tissues : heart, liver, and skeletal muscle. Henion and Sutherland (1957) demonstrated immunological differences between liver and skeletal muscle phosphorylases. Examples of immunological differences among analogous enzymes presented above are but a portion of many examples in the literature. These demonstrate subtle biochemical differences between the host and the parasite or even between different organs within the same species. The nature of these differences is not yet well understood. Further studies on these lines might be of value in finding chemical agents which can selectively inhibit or stimulate a certain enzyme without affecting analogous enzymes.

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VII. Effect of Anthelmintic Agents on Carbohydrate Metabolism

A. GENERAL Chemotherapy, as established by Ehrlich in the early part of this century, is based on the selective inactivation of certain receptors in the parasites without affecting the host cells. Guided by this principle many workers in the field have been approaching the problem of chemotherapy of parasitic organisms through a search for differences between the metabolism of the parasite and that of the host. Such investigations may uncover new receptors in the parasite which can be affected by drugs without any damage to the host. In addition to studies on the physiology and biochemistry of parasitic helminths attempts have been made to locate mechanisms of action of known chemotherapeutic agents a t cellular and subcellular levels. The evidence available today from studies on helminths as well as on bacteria and protozoa strongly indicates that a large number of these agents act on enzyme systems. Because parasitic helminths are dependent on carbohydrate metabolism for their survival, emphasis has been placed on discovering the mechanism of action of anthelmintic agents on the individual enzymes of the parasite carbohydrate metabolism. Studies on the mechanism of action of known chcmotherapeutic agents might reveal new enzymes which are more vulnerable to inhibition by drugs. It is with this end in view that the activity of the following chemical agents has been investigated. Several subjects which are discussed here have been previously reviewed by Bueding and Swartzwelder (1957) and Bueding (1962b). B. THEANTISCHISTOSOMAL ACTIONOF ANTIMONIALS In spite of recent intensive research, trivalent antimonials have remained the most effective chemotherapeutic agents against schistosomiasis. Bueding (1950) reported that glycolysis in intact worms is inhibited by trivalent antimonials, stibophen (IV) and antimony potassium tartrate (tartar emetic) (V). Attempts were made by Mansour and Bueding

o=c-0 Na03S y 7\4 o > / O q s\o 3 N a

0 NaO S0,Na

S0,Na

(Iv1

I \ HC-0-Sb HC-0 I / I COOK

(V1

(1954) to elucidate the mechanism of action of these agents on the glycolytic enzymes. The effect of antimonials on lactic acid production by

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cell-free extracts from Schistosoma mansoni was tested (Mansour and Bueding, 1954). Lactic acid production was markedly reduced in the presence of low concentrations of antimonials when glucose or F-6-P was the substrate. Both tartar emetic and stibophen had little effect on the parasites' hexokinase and no effect on phosphohexose isomerase. Thus the first two steps in the Meyerhof scheme for glycolysis were not affected. When FDP was used as the substrate, lactic acid production was not affected even by high concentrations of antimonial. These experiments indicated that inhibition of glycolysis by antimonials is brought about by blocking the formation of FDP from F-6-P catalyzed by the action of phosphofructokinase. I n confirmation of this i t was found that phosphofructokinase from the schistosome was inhibited markedly by low concentrations of trivalent antimonials. On the other hand, mammalian phosphofructokinase was inhibited only a t concentrations 80 times higher than those necessary to inhibit the worm enzyme (Mansour and Bueding, 1954). Thus it appears that the antimonials inhibit selectively schistosome phosphofructokinase without affecting the host enzyme. Subsequently Bueding and Mansour (1957) confirmed these results by demonstrating that the inhibitory effect of antimonials on lactic acid production from glucose by schistosome extracts is abolished by the addition of purified mammalian phosphofructokinase in excess. Furthermore, exposure of schistosomes to low concentrations of antimonials or administration of subcurative doses of antimonials to infected mice resulted in accumulation of F-6-P and in a reduction in the concentration of FDP in the worms, indicating that the activity of phosphofructokinase within the intact worms was inhibited. Reduction in the concentration of FDP in the worms reduces the activity of the enzyme aldolase, which has a high requirement for this substrate. The final result is an inhibition of glycolysis.

C. THEBIOCHEMICAL EFFECTS OF DITHIAZANINE A different facet of carbohydrate metabolism has been reported to be inhibited by dithiazanine. This is 3,3'-diethylthiadicarbocyanine iodide

(VI)

(VI) which was introduced in recent years for the treatment of several intestinal nematode infections (Swartzwelder et al., 1957). Studies on the biochemical effects of dithiazanine against Trichuris vulpis, which is

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very susceptible to the chemotherapeutic action of the dye, was reported by Bueding et al. (1961). Their studies revealed that the parasites can be maintained in an atmosphere containing CO, in nitrogen for a longer period of time than in air or a mixture of CO, and oxygen, indicating that these worms do not depend on oxidative metabolism for survival. Glucose utilization by these organisms under anaerobic conditions was high. Approximately 50% of the utilized glucose is accounted for by lactic acid, propionic, n-valeric acid, carbon dioxide, and small quantities of formic acid and n-butyric acids, Dithiazanine in concentrations which do not affect the motility of Trichuris vulpis resulted in inhibition of the uptake of glucose by the parasite. This effect was found to be irreversible; when the worms, after exposure to the drug, were transferred to a dithiazaninefree medium the inhibition of glucose uptake persisted. Reduction of glucose uptake by dithiazanine was associated with a corresponding decrease in the amount of volatile fatty acids produced. The concentration of free glucose in the dithiazanine-treated worms was found to be markedly reduced. This effect was interpreted to indicate that the inhibitory action of the drug on glucose uptake by the worm is not due to inhibition of glucose utilization by the cell but rather due to a decrease in the glucose entering the ccll. An inhibition in one of the reactions essential for intracellular glucose utilization might be expected to result in an increase in the intracellular concentration of free glucose. Bueding et al. (1961) concluded that the effect of dithiazanine could be due to its interference with the transport of glucose in the worms. This would result in a reduction in the carbohydrate reserve stores and inability to generate energyrich phosphate bonds. This was supported by the finding that the levels of ATP and endogenous glycogen in the parasites were markedly decreased.

D. THE ANTISCHISTOSOMAL ACTIONOF BENZYLIC DIAMINM Although none of the beneylic diamines is used for clinical treatment of schistosomiasis some members of this group were reported to have antischistosomal effects in vitro (Bueding, 19624. The most active compound was l16-bis- [ p - (N-methylaminomethyl)1-phenoxyhexane (VII).

(VII 1

Reduction in worm motility produced by these compounds was preceded by an inhibition of glucose utilization and lactic acid production as well as an increase in the rate of glycogenolysis of the worms (Bueding, 1 9 6 2 ~ ) .

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Concentrations of diamine considerably higher than those which produced inhibition of glucose uptake in the intact organism did not affect the rate of glycolysis of cell-free extracts of schistosomes. Furthermore, the activities of hexokinase, of phosphorylase, of adenosinetriphosphatase, or of phosphoglucomutase were not changed. These results indicated that the diamines exert no direct effect on enzymes involved in glycolysis and might interfere with the active transport of glucose into the worm. The increase in glycogenolysis could be due to a reduction in the availability of dextrose from an exogenous source (Bueding, 1 9 6 2 ~ ) . Exposure of schistosomes to low concentrations of a benzylic diamine and the organic antimonial, stibophen, resulted in a much greater reduction of survival than with the same concentration of each compound alone (Bueding, 1 9 6 2 ~ ) .It appears then that Schistosomu mansoni is more vulnerable to inhibition of two phases of its carbohydrate metabolism, namely, phosphofructokinase inhibition by antimonials and the mechanism for glucose uptake by the diamine, than to interference a t only one of these levels.

E. THEANTIFILARIAL ACTIONOF CYANINE DYES A group of cyanine dyes were reported to have high activity both in vitro and in vivo against a filarial worm which lives in the pleural cavity of the wild cotton rat Litm.osoides carinii (Welch et al., 1947; H. N. Wright et al., 1948; Peters et al., 1949). Unlike many related organisms Litomosoides carinii is dependent for its survival on oxidative metabolism (Bueding, 194913). The cyanine dyes in concentrations as low as 5 X M inhibit the oxidative metabolism of these worms. The decrease in oxidative metabolism was associated with a compensatory increase in aerobic glycolysis and with decreased rates of acetate formation and polysaccharide synthesis. The same metabolic changes were observed in filariae removed from cotton rats to which subcurative doses of a cyanine dye had been administered. This indicates that the effect of these compounds on the metabolism of the filarial worms can be demonstrated in vivo as well as in vitro. It is probable, therefore, that these compounds exert their chemotherapeutic effect through inhibition of enzyme systems concerned with oxidative metabolism. The cyanine dyes in concentrations 500-1000 times higher than those required to inhibit the respiration of Litmosoides carinii have no effect on the oxygen uptake of slices and homogenates of mammalian tissues. Thus, the selective toxicity of the cyanine dye against filariae can be explained by the finding that it inhibits in the worms an enzyme system that plays no role or one of only minor importance in mammalian tissues (Bueding, 1949b). I n contrast to their effect against Litmosoides carinii the cyanine dyes

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have no effect against mature Schistosoma mansoni (Bueding et al., 1953) although they depress the respiration of schistosomes in vitro and in vivo (Bueding, 1950; Bueding et al., 1953). This is in line with Bueding’s observation that Schistosoma mansoni is not dependent for its survival on an oxidative metabolism. VIII. Conclusion

The facts which have been discussed above indicate important biochemical and physiological differences between the host and parasitic helminths on one hand, and among different parasites on the other. This has been demonstrated a t various levels: on the intact organisms, on cellfree extracts, and on isolated enzyme systems. It has been observed that many of the chemotherapeutic agents which are now in use against helminths owe their effect to a selective action on certain physiological or biochemical processes in the parasite. Advances in the chemotherapy of helminthiasis will depend greatly on more knowledge concerning the physiology, biochemistry, and pharmacology of these animals. The presence of the same metabolic pathway or the same physiological process in the host and parasite does not rule out differences between the two. Such differences have been shown to occur even among enzymes catalyzing the same reactions. Further studies along these lines will no doubt lead to the development of more effective and less toxic chemotherapeutic agents. Information obtained from these studies may also be highly important in elucidating some of the regulatory mechanisms of biochcmical processes in higher organisms. ACICNOWLEDOMENT Some of the work of the author, reviewed above, was supported by Public Health Service Research Grant AI-04214 from the National Institute of Allergy and Infectious Diseases, and Research Career Development Award GM-K3-3848 from the Division of General Medical Sciences.

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The Adrenergic System and Sympathomimetic Amines E. MABLEY Institute of Psychiatry. Maudsley Hospital. London. England

I . Introduction . . . . . . . . . . . . . . . I1. The Adrenergic System . . . . . . . . . . . . A . Adrenergic Neurone in Peripheral Autonomic Nerves . . . . B. C h r o m a h Cells . . . . . . . . . . . . . C. The Noradrenaline Store . . . . . . . . . . . D . Cholinergic Fibers in Sympathetic Nerves . . . . . . . I11. Adrenergic Receptors . . . . . . . . . . . . IV. Formation of Catecholamines . . . . . . . . . . V. Preganglionic Nerves and Sympathin Secretion: Adrenal Gland . . A. Sympathin-Secreting Cells . . . . . . . . . . B Nerve Supply . . . . . . . . . . . . . . C. Resting Secretion of Amines . . . . . . . . . . D. Discharge of Adrenal Sympathin . . . . . . . . . E. Splanchnic Nerve Excitation . . . . . . . . . . F. Extirpation of the Adrenal Glands . . . . . . . . . G . Central Control of Adrenal Medullary Secretion . . . . . H. Adrenal Medullary Secretion and the Central Nervous System: . . . . . . . . . . . . . . Awareness \? . Postganglionic I Sympathetic Nerves and Sympathin Secretion : The Spleen . . . . . . . . . . . . . . . . A . Liberation of Transmitter . . . . . . . . . . . B. Uptake of Noradrenaline on Splenic Receptors . . . . . . VII. Peripheral Action of Sympathomimetic Amines: Structure-Activity Studies . . . . . . . . . . . . . . . . A . No Differentiation between Adrenergic Receptor and Adrenergic Neuron . . . . . . . . . . . . . . . B. Differentiation between Adrenergic Receptor and Adrenergic Neuron . . . . . . . . . . . . . . . C . Differentiation between Adrenergic Receptor, Adrenergic Neuron and/or Chromaffin Cell . . . . . . . . . . . VIII . Sympathomimetic Amines and the Central Nervous System . . . A . Sympathin in Brain . . . . . . . . . . . . B. Action on Behavior and Cerebral Electrical Activity . . . . C . Direct or Indirect Action of Catecholamines . . . . . . D . Intraventricular and Intracisternal Injection of Drugs . . . . E . Amino Acid Precursors of Catecholamines . . . . . . . F. Action of Sympathomimetic Amines in Immature animals . . . G. Iontophoretic Application of Drugs to Neurons . . . . . H. Quantification of the Effects on the Central Nervous System . . I. Effects in Groups of Animals . . . . . . . . . . J. Side Effects in Man . . . . . . . . . . . .

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IX. Blockade of Adrenergic Neuron and Receptor A. Blockade of Adrenergic Receptor . . B. Blockade of Adrenergic Neuron . . X. Inactivation of Amines . . . . . A. Dcamination . . . . . . . B. O-Methylation . . . . . . C. N-Demethylation . . . . . . D. O-Dcmethylation . . . . . . E. Hydroxylation . . . . . . F. Binding of Amines . . . . . XI. Conclusion . . . . . . . . References . . . . . . . .

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I. Introduction

The adrenergic system consists of adrenergic neurons, chromaffin cells, and adrenergic receptors. Noradrenaline is liberated a t postganglionic sympathetic nerve terminals; in the adrenal gland the secretor cells which form adrenaline and noradrenaline are homologous with adrenergic neurons. Adrenaline acts mainly as a hormone, noradrenaline as a hormone, as a transmitter of nerve impulses, and as an intermediate in adrenaline formation. Adrenaline and noradrenaline (sympathin) belong to a series of chemical compounds which simulate the effects of sympathetic nerve excitation not only with varying intensity but with varying precision. These are the sympathomimetic amines (Barger and Dale, 1910). The sympathomimetic amines, depending on chemical structure, act either directly on the adrenergic receptor or rely for their effect on the integrity of the postganglionic adrenergic neuron and chromaffin cells. II. The Adrenergic System

A. ADRENERGIC NEURONS IN PERIPHERAL AUTONOMIC NERVES The first indication of the presence of an adrenaline-like substance in adrenergic nerves was obtained by Gaddum and Khayal (quoted by Euler, 1956) ; the substance was released into the surrounding fluid after excitation of an isolated portion of the splanchnic nerve. Lissak (1939) demonstrated that adrenergic nerve extracts have adrenaline-likc properties ; activity disappears when the nerves degenerate (Cannon and Lissak, 1939; Goodall, 1951). The activity of extracts is due chiefly to noradrenaline, although minute quantities of adrenaline are present (Euler, 1949). The noradrenaline content varies in different nerves, the relative noradrenaline activity per unit weight being closely related to the amount of adrenergic tissue present. There is a small amount of nor-

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adrenaline in the vagus and the short ciliary nerves suggesting the presence of adrenergic neurons; in fact, sympathetic fibers run in the cervical vagus (Jones, 1932; Agostoni e t al., 1957). Dopamine is found in sympathetic nerves and ganglia (Schiimann, 1956; Euler, 1958). I n extracts of the sympathetic nerves of pulmonary and splenic vessels the catecholamines are almost exclusively dopamine and noradrenaline (Euler and Lishajko, 1958). Dopamine is of special interest as the precursor of noradrenaline. Some of the evidence for the presence of adrenaline in nerve extracts is open to the criticism that dopamine might have interfered with the bioassays (Hoke, 1959), but this criticism does not apply to experiments in which the amines were separated by chromatography and assayed on the rat uterus (Muscholl and Vogt, 1958; Vogt, 1959; Muscholl, 1959). Noradrenaline, together with a variable but small amount of adrenaline, is released after adrenergic nerve excitation (Peart, 1949 ; Mann and West, 1951; Outschoorn, 1952; Mirkin and Bonnycastle, 1954) ; the adrenaline may, however, come from chromaffin cells, (Euler, 1956). Prolonged stimulation does not alter the noradrenaline content of nerves (Luco and Goni, 1948) or ganglia (Vogt, 1954). The terminal parts of the adrenergic fibers are difficult to study, but there are reasons for believing that the sympathin content is greatly increased in the region of nerve endings. The ox splenic nerve contains about 15 pg noradrenaline/gm whereas the spleen contains about 3 pg/ gm. The 5: 1 ratio implies that the terminal paths of the nerve within the spleen contain far larger amounts of sympathin per unit weight than the preterminal axons. Assuming a mass of nerve endings having l/lO,OOO1/1000 of the organ weight, this would give figures of 3-30 pg amine/ mg of nerve terminals (Euler, 1956). It is uncertain whether the catecholamines are stored in nerve endings or in the associated chromaffin cells. If the postganglionic sympathetic nerves are cut, the concentration of noradrenaline in the tissue declines as the nerve degenerates whereas that of adrenaline does not (Euler, 1961). This suggests that noradrenaline is associated with sympathetic nerve endings and adrenaline with some other structure, possibly the chromaffin cells. B. CHROMAFFIN CELLS Chromaffin cells and sympathetic nerves are related developmentally for both arise from ectoderm (J. D. Boyd, 1961). Chromaffin tissue is widely distributed. Chromaffin granules are present in the adrenal medullary cells of all vertebrates (J. D. Boyd, 1961), in sympathetic ganglia and nerve (Kohn, 1903), in the cat’s mesenteric and solar ganglia

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(Muscholl and Vogt, 1958), in rabbit arterial wall (Burn and Rand, 1957a), in the cat’s arrector pili muscles and nictitating membrane (Burn et al., 1959), in human skin (Adams-Ray and Nordenstam, 1956; Phillips et al., 1960; Coupland and Heath, 1961), and in the dog’s hypothalamus (Chruiciel, 1961). I n sympathetic nerves the noradrenaline is stored in granules (Schiimann, 1958; Euler, 1958) the adenosine triphosphate (ATP) content of which has a definite relation to the amount of amine present (Schiimann, 1958). Presumably each ATP molecule with its four negative charges holds four amine molecules by electrostatic forces (Hillarp, 1961). I n sympathetic nerves about half the amine is dopamine and half noradrenaline, the noradrenaline being held within chromaffin granules and the dopamine outside (Schiimann, 1958). All chromaffin cells may not necessarily be associated with nerve fibers, for in the noninnervated hagfish heart (Augustinsson e t al., 1956) there are granules resembling adrenal medullary cells (Ostlund et al., 1960). The granules differ sufficiently to make generalizations hazardous. Adrenal medullary and intestinal mucosal granules are equally sensitive to procedures that disrupt membranes, whereas granules from adrenergic fibers are relatively resistant t o freezing, thawing, and to surface active agents (Euler and Lishajko, 1961). This would imply a different method of release under physiological conditions of amines from adrenal medullary granules and from those a t nerve endings. C. THENORADRENALINE STORE Organs innervated by adrenergic nerves contain noradrenaline in variable amounts although the quantity is remarkably constant for any one organ, indicating stores of fixed capacity. Although i t is recognized that noradrenaline may be stored in granules, it has now been suggested that adrenergic fibers take up noradrenaline from the blood and hold it in such a way that it can be subsequently released. There may be several stages between the uptake of noradrenaline and its incorporation into the store; incorporation into granules may indeed not occur, for that present may be the result of synthesis only, The store is not inert and differs from noradrenaline inactivated by binding on receptors. There may be two components or pools to the store as suggested by Hillarp (1961) for the adrenal medulla, one of which is bound in the granules and the other extragranular, accounting for 80% of the amine, and readily mobilized. The amine bound within the granules would be nondiffusible (Hillarp, 1961). The two pools would be in equilibrium but distinct. That radioactive noradrenaline does not readily mix with the large amount of noradrenaline in tissues favors this view (Dengler e t al., 1961a). Presumably the whole store would be separated from the enzyme monoamine

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oxidase by an intracellular membrane which also keeps the amine unavailable to receptor sites (Brodie and Costa, 1962). The noradrenaline store may be in the adrenergic neurons (Euler, 1961), in the chromaffin cells, or in both; the size of the store determines the response to sympathetic stimulation. There are several facets of evidence suggesting noradrenaline uptake by the store. (1) Noradrenaline disappears from postganglionic sympathetic fibers after section and degeneration (Euler and Purkhold, 1951; Goodall, 1951; Burn and Rand, 1959) but is unaffected by preganglionic section; it reappears on regeneration of the postganglionic fibers (Goodall, 1951). The effects of amines which are presumed to be mediated through noradrenaline release, such as tyramine, cannot be elicited on denervated tissues and are not restored by noradrenaline infusion (Burn and Rand, 1960). This suggests that the store resides in the postganglionic segment of adrenergic nerves and that uptake depends upon its viability. (2) Reserpine depletes tissue stores of noradrenaline (Bertler et al., 1956; Burn and Rand, 1957a, 1958a, 1959; Carlsson and Hillarp, 1956; Holzbauer and Vogt, 1956; Pletscher et al., 1955). Whereas the chromaffin granules disappear from degenerating nerve, a “ghost” of the granule remains after reserpine treatment. The effects of amines mediated through noradrenaline release are lost on reserpinized tissues (Burn and Rand, 1958b, 1959) but restored by noradrenaline infusion (Burn and Rand, 1958b, 1960), although reserpine treatment may impair the noradrenaline storage process (Pennefather and Rand, 1960; Brodie and Costa, 1962). I n reserpine-treated cats dopamine infusion restores the pressor and mydriatic action of tyramine (Burn and Rand, 1960). Other noradrenaline precursors are effective; 50 pg dopamine, 1 mg L-dopa, 2 mg m-tyrosine, or 25 mg phenylalanine infused into the reserpinized rat restores the pressor action of tyramine (Burn and Rand, 1960). Tissues innervated by sympathetic nerves appear, therefore, to have cells which take up noradrenaline from the blood. Isolated tissues also take up noradrenaline (Gillespie and MacKenna, 1959; Azarnoff and Burn, 1961; Hukovi6, 1961). (3) Even in normal cats, the effects of sympathetic stimulation are increased by noradrenaline. In the dog’s perfused hindleg, tyramine caused constriction and reduction of venous outflow; after an infusion of noradrenaline the effect of tyramine was much enhanced (Burn and Rand, 1960). (4) The crucial evidence turns on direct measurement of noradrenaline uptake by tissues. Thus, after the intraperitoneal injections of large doses of noradrenaline and adrenaline in the dog there was a twelvefold increase in heart noradrenaline and even greater increase of adrenaline

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(Raab and Gigee, 1955). Euler (1956) gave much smaller quantities of amine but did not find any uptake of amine in the heart, spleen, liver, kidney, and skeletal muscle. Pennefather and Rand (1960) infused noradrenaline into spinal eviscerated cats. At the beginning of the experiment they removed one kidney and one uterine horn. The extractable noradrenaline was two- to threefold greater in the remaining kidney and uterine horn after the infusion in comparison to the control tissues; the increase could not be attributed to the blood content of the organ. Whitby et aZ. (1960) injected d-P-H3-noradrenaline into cats. The H3-noradrenaline was taken up by the adrenal gland, heart, and spleen; small amounts were taken up by the liver and skeletal muscle. There is considerable evidence then for noradrenaline uptake by tissues. If uptake is an active mechanism then it should be susceptible to metabolic or pharmacological interference. This is so. The prior administration of amphetamine, chlorpromaeine, cocaine, guanethidine, imipramine, phenoxybenzamine, reserpine, and tyramine markedly diminished the amount of H3-noradrenaline found in tissues after intravenous infusion (Axelrod et al., 1961; Hertting et aZ., 1961a, 1961b). Imipramine and cocaine may compete with the storage process for noradrenaline (Brodie and Costa, 1962). With in vitro experiments, H3-noradrenaline uptake by tissues was reduced in addition by bretylium, dichloroisopropyl noradrenaline (DCI) , ephedrine, ouabain, Recanescine, rescinnamine, reserpine, thyroxine, and tyramine (Dengler e t al., 1961b, 1962). If the action of amines like tyramine is mediated by a release of noradrenaline from tissue stores, the question arises as to whether the released noradrenaline circulates in the blood. In fact, not only is there no evidence for this (Vane, 1961), but there is some indication that it is adrenaline and not noradrenaline that is released locally (West, 1961). One of the features that distinguishes the amphetamine-like amines from the catecholamines is that tachyphylaxis develops on repeated administration of the former. Tachyphylaxis to the pressor effects of phenylalkylamines is apparently related to the exhaustion of available noradrenaline from the stores in adrenergic nerve fibers (Cowan et al., 1961; Potter et al., 1962). D. CHOLINERGIC FIBERS IN SYMPATHETIC NERVES The classic idea of adrenergic transmission was that of a nerve impulse liberating adrenaline from the nerve endings close to the site of action (Elliott, 1904). The transmitter proved subsequently to be noradrenaline (Euler, 1948; Peart, 1949). The concept that postganglionic sympathetic fibers might in some

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cases be cholinergic is not new, Cholinergic fibers occur in Sympathetic nerves to the vessels of the tongue (Euler and Gaddum, 1931b), to the sweat glands (Dale and Feldberg, 1934), to the vessels of the dog’s hindleg (Bulbring and Burn, 1935), to the cat’s nictitating membrane (Bacq and Fredericq, 1935) and heart (Folkow e t al., 1948), and the dog’s uterus (Sherif, 1935). From the pattern of distribution of acetylcholinesterase in adrenergic nerves, Koelle (1961) concluded that acetylcholine may be involved in the transmission of a proportion of sympathetic nerve endings. Many sympathomimetic effects are produced in the body by acetylcholine and by nicotine in the presence of atropine in circumstances in which they act beyond the location of the sympathetic ganglia. I n the isolated perfused rabbit ear, stimulation of the sympathetic postganglionic fibers constricts the vessels; so does the injection of nicotine into the perfusing fluid, or when, in the presence of atropine, acetylcholine is injected. There are striking similarities in the effect of acetylcholine and of sympathetic nerve excitation on the isolated atria and colon of the rabbit and of the pilomotor response of the cat’s tail. The effects cannot be obtained after pretreatment with reserpine (Burn, 1961; Burn and Rand, 1958b, 1960; Burn et al., 1959; Gillespie and MacKenna, 1959). The idea of an underlying cholinergic mechanism to include all postganglionic sympathetic fibers was proposed by Burn (1961) and Burn and Rand (1962). Acetylcholine liberated from cholingeric nerve endings would release sympathetic transmitter from a local tissue store which acts on the effector. Bretylium blocked the action of acetylcholine and of sympathetic nerve excitation but not of tyramine on the isolated rabbit atria (Hukovi6, 1960; Burn, 1961). Bretylium is a quaternary ammonium salt and so more likely to affect acetylcholine than noradrenaline transmission. Burn suggested that just as acetylcholine is specifically blocked by atropine a t receptors associated with parasympathetic nerve endings, by tubocurarine a t the neuromuscular junction, and by hexamethonium a t ganglia, so it is blocked a t sympathetic postganglionic terminals by bretylium ; since all postgangIionic sympathetic nerves are blocked by bretylium, they must all mediate their effects through acetylcholine release. Burn and Froede (1963) noted that of the nine compounds now shown to block responses to sympathetic postganglionic nerve stimulation, seven were “onium” compounds related in structure or action, or in both, to acetylcholine but not to noradrenaline. That hemicholinium blocks transmission in some isolated nervesmooth muscle preparations (Chang and Rand, 1960) has been construed as evidence that sympathetic nerves are cholinergic. This is because

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hemicholinium interferes with acetylcholine synthesis (MacIntosh et al., 1956) by preventing access of choline to choline acetylase (Gardiner, 1961). As Vane (1962) points out, general acceptance of the new hypothesis will depend to a large extent on further research on the specificity of both bretylium and hemicholinium and anatomical analysis of the nerve endings, He suggests that the presence of a small proportion of cholinergic fibers in sympathetic nerves might also explain the results of Burn and Rand. Holmstedt and Sjoqvist (1959) found that relatively few cells of the cat’s sympathetic ganglia were cholinergic in the sense that they contained cholinesterase. This would endorse the view of a minority of cholinergic fibers in postganglionic sympathetic nerves, A number of observations do not accord with the idea that all sympathetic nerves have a n underlying cholinergic mechanism. When the cat’s nictitating membrane was isolated together with its nerve, there was no evidence that acetylcholine was involved in transmission from nerve to smooth muscle (Gardiner and Thompson, 1961) ; similar conclusions were made from in vivo experiments with the nictitating membrane (Cervoni et al., 1956). If acetylcholine is involved in the transmission between sympathetic nerve and effector it should be possible to demonstrate the presence of acetylcholinesterase in the smooth muscle of the nictitating membrane. Cholinesterase was found only in the Harderian gland; there was none in the smooth muscle cells of the membrane nor in the great majority of the nerve fibers supplying the muscle (Hellman and Thompson, 1961; Gardiner et al.,1962). Whereas Chang and Rand (1960) tested the effect of hemicholinium on tissues most of which contained cholinergic and adrenergic fibers in their nerve supply, Gardiner and Thompson (1961) tested the effect of hemicholinium on the purely adrenergic isolated nictitating membrane preparation ; even with high doses of hemicholinium they were unable to affect transmission. 111. Adrenergic Receptors

The chemical group in the tissue with which a single molecule of drug combines is called a receptor. Although the idea of “receptors” was introduced by Ehrlich, the notion that adrenaline reacts with receptors was due to Langley (1906). He postulated a “receptive substance” interposed between nerve and muscle on which adrenaline acts. Dale (1906) differentiated two types of adrenergic receptor; one type on which adrenaline gave excitatory responses was “paralyzed” by ergot alkaloids, whereas the other on which adrenaline elicited inhibitory responses was not. Ahlquist (1948) defined adrenotropic receptors as hypothetical structures or systems located in or near the muscle or gland cells affected

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by adrenaline. Although receptors may be closely related to enzymes there are differences, partly due to the fact that the receptor has apparently a more or less rigid geometrical structure (Schild, 1962). Until recently receptors were regarded as permanent entities but it now argued that they have a limited life-span, and are being continuously produced (Miledi, 1962). Quantitative theories of the action of drugs of the type acetylcholine and atropine rest chiefly on the classic work of Clark (1926, 1933) and Gaddum (1926, 1937), who suggested that the combination of drugs with specific receptor groups may be described by the mass action relations derived from the Langmuir (1918) adsorption isotherm or from the Michaelis-Menten assumptions. The concept of drug-receptor interactions developed by Clark (1937) for interpreting the quantitative relation between concentration of applied drug and its action on a tissue led to equations analogous to those relating the velocity of an enzyme reaction to concentrations of substrate. An elegant alternative was proposed by Paton (1961b), who put forward a theory of drug action based on the rate of drug-receptor combination. Specificity is a property of receptors; i t is also a property of proteins, particularly enzymes, and the question arises whether the receptor is an enzyme. One question must be asked before interpreting receptor properties (Belleau, 1961) : Is it a prerequisite for the triggering of a response that the agonist molecule should play the role of substrate for receptor sites, or is the response comparable t o a molecular interaction triggered through electrostatic field effects and van der Waals’ forces? Evidence that noradrenaline was not a substrate for the receptor was offered by Belleau and Pindell (quoted by Belleau, 1961). Since simple agonists such as /3-phenylethylamine exist in the ammonium form a t physiological pH and since no other polar or ionizable groups are present in their molecules, the triggering mechanism must in all probability be the result of an electrostatic interaction a t the receptor level with a group of opposite charge. Belleau suggested that an ion-pair association occurred leading to charge neutralization of an anionic site, the positively charged nitrogen approaching the negatively charged site of the receptor. The importance of the negatively charged head was evident from an experiment of Briicke e t al. (1959). They prepared a substituted noradrenaline, in which the nitrogen was part of a glycine residue. The compound had no activity, but when the carboxyl group was esterified so that the side chain again carried a basic group, activity was regained. By analogy with the adrenal medulla, where catecholamines are released from a site containing ATP, it is tempting to postulate that the amine would interact with ATP a t the receptor. A phosphate anion was

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therefore likely to be the chemical species initially interacting with the amine and a phosphorylated protein would be the adrenergic receptor. It has also been suggested that an iron-containing protein is a component of the adrenergic receptor (Imaizumi, 1961). Compounds with a small cationic head, for example, P-plienylethylamine, had strong excitatory activity, whereas hydroxyl groups in the 3, 4-position on the ring conferred inhibitory activity. Gencrally speaking, a substance such as p-phenylethylamine, which reproduces the backbone of noradrenaline, tended to be more active than other substances, pointing to a special affinity of a specific receptor sitc for aromatic rings. The anchoring sites of adrenaline would be a t the nitrogen atom, the phenyl ring, and a t points near the 3,4-hydroxy groups (Belleau, 1958). Blaschko (1950) had suggested that tissue receptors for adrenaline had three anchorages: one each for the catechol, the p-hydroxyl, and the NH.CH, group. Whercas Belleau’s ideas apply to all sympathomimetic amines, several workers have confined themselves to the catecholamines. Catecholamines have varied actions in the body: they stimulate the heart, dilate the bronchi, contract vascular smooth muscle, and inhibit intestinal smooth muscle, as well as alter metabolism. There appear to be a t least two classes of receptors for catecholamines, activation of the one resulting in excitation and of the other, in inhibition of the effector cells (Ahlquist, 1948). There were exceptions, for Ahlquist (1948) showed that these amines might excite one receptor and inhibit another. Of six catecholamines tested, there was one order of potency (1, 2, 3, 4, 5, 6) for vasoconstriction, excitation of the uterus and ureters, contraction of the nictitating membrane, dilatation of the pupil, and inhibition of the gut, the effect a t &-receptors. I n contrast, the same amines had an entirely different order of potency (2, 4, 6, 5, 3, 1 ) for vasodilatation, inhibition of the uterus, and myocardial stimulation, the effect a t P-receptors. Most of the excitatory effects were blocked by antagonists such as ergotoxine and phenoxybeneamine ; the inhibitory and metabolic effects and the action on the heart were not. Lands (1952) objected to certain aspects of Ahlquist’s classification and preferred to regard adrenergic receptors in the heart as undifferentiated. This dichotomy between the effects antagonized by blocking agents and those that were not, further disposed to the idea that there were two types of receptor. Receptors blocked by ergotoxine, Dibenamine, and phenoxybenzamine were the a-receptors and those which were not blocked, the /&receptors. Compounds which selectively block &receptors have been recently discovered (Powell and Slater, 1958; Maycr and Moran, 1960; Ariens, 1961). A number are similar in structure to the

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catecholamines, but the ring hydroxyls have been replaced by chlorine atoms; another P-blocker is novel in that it is a naphthylamine (Black and Stephenson, 1962). Because the inhibitory effects of catecholamines on intestinal smooth muscle were not completely blocked by either type of antagonist, another receptor (&receptor) was proposed (Furchgott, 1959). However, a combination of both types of antagonists gives complete blocking, indicating that the inhibitory action of catecholamines on intestinal smooth muscle is mediated through and P-receptors (Ahlquist and Levy, 1959; Furchgott, 1961). According to this concept, noradrenaline acts mainly on a-receptors, isoprenaline mainly on /I-receptors, and adrenaline on both. By using the two types of antagonist and by comparing the activity of noradrenaline with that of isoprenaline, the distribution of a- and preceptors has been plotted. Folkow (1961) studied the effects of the catechol amines on consecutive vascular sections. The a-receptors were widespread in resistance and capacitance vessels, whereas P-receptors were concentrated on the resistance side of vessels of skeletal muscle. Ariens (1961) investigated the effects of substituents on the phenyl radical or on the amino group of catecholamines and concluded that the intrinsic activity for action of catecholamines on a-receptors was especially related to the interaction of the amino group and a-receptor, while the action on P-receptors may be especially related to the interaction of the catechol group and &receptor. The primary action of catecholamines on many tissues is to promote the formation from ATP of a cyclic nucleotide, adenosine 3’,5’-phosphate (Sutherland and Rall, 1961). The enzyme which converts ATP to cyclic adenylate requires a bivalent metal such as magnesium; Belleau (1961) suggested that the hydroxyl groups of the catechol nucleus combine with a bivalent metal ion attached to the enzyme. The cyclic adenylate stimulates the formation of active phosphorylase which accelerates glycogenolysis. ATP is thus intimately connected with catecholamines not only in their storage but also their effects on metabolism It is possible that this cyclase system is the P-receptor, particularly as isoprenaline is the most effective of the catecholamines upon the system and adrenaline is generally more effective than noradrenaline. The cyclase system may be in the cell membrane or inside the cell (Sutherland, 1961). The difference between the actions of isoprenaline, adrenaline, and noradrenaline may depend on the ease with which they penetrate cell membranes. The adrenergic receptor may in fact be located intracellularly or in the cell membrane. The strongest evidence for an intracellular location of adrenergic receptors is the glycogenolytic effect of adrenaline and closely related sympathomimetic amines in liver, skeletal, cardiac, and (Y-

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certain smooth muscles (Furchgott, 1959). Boeler (1940) pointed to the variable and often diphasic action of adrenaline on visceral smooth muscle. Using electrophysiological methods, adrenaline can be shown to have two actions on intestinal smooth muscle (Biilbring, 1961), and these may be related to their a- and p-effects. There is a direct action on the membrane, with probably an increase in permeability to one or several ions which leads to depolarization, making the membrane less stable and more excitable. There is also a metabolic action indirectly affecting the membrane, with increase in phosphorylase activity. The increased energy made available can be used either for the contractile mechanism, or to stabilize the membrane and render it less excitable. The hypothesis implies that in smooth muscle catecholamines induce both a- and p-effects simultaneously and the observed effect is the resultant of two opposing actions. When contraction is produced the direct action on membrane permeability predominates; for relaxation, the metabolic effect is predominant. This is a far-reaching and valuable interpretation and while not contradicting Ahlquist’s (1948) notion of receptors is more easily linked than his with the generalizations on structure and activity for sympathomimetic amines by AriEns (1961) and Belleau (1961). A complication is that adrenaline inhibits muscles which have been completely depolarieed by immersion in high concentrations of potassium salts (Schild, 1961). Experiments with antagonists indicated that adrenaline was acting on the same receptors in normal and depolarieed muscle, so that in depolarized muscle not even an indirect action on the membrane could account for the inhibition. Are the receptors for sympathomimetic amines in the central nervous system similar to those in peripheral tissues? If structure-activity studies of the sympathomimetic amines on peripheral and central receptors can be intercorrelated and the activity on central receptors of an amine relative to others of similar structure parallels its activity on peripheral receptors, there seems reasonable grounds for a t least likening the geometry of the one receptor with that of the other, although combination with the different receptors might involve different mechanisms. The concept of intracranial receptors has been reviewed by Winterstein (1961). H e applied the term to “specific nervous formations especially adapted to receive chemical stimuli and to transmit these excitations to other centres, in the same way as the nerve endings of the sensory organs are adapted to their specific stimuli.” Interpreted in its widest sense, these “special receptors” must be in continuity with, or accessible from, the cerebrospinal fluid if the soporific action of catecholamines on intracisternal injection (Leimdorfer, 1950) is to be explained. Receptors may be found a t various levels of the central nervous system

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and Feldberg (1958) designated the periventricular gray matter as an as yet scarcely explored area of high pharmacological activity. Receptors therein would be accessible to drugs given by intraventricular injection. Bradley and Elkes (1953) likened the behavioral and electrocortical arousal produced by amphetamine to that evoked by stimulation of the reticular-activating system. As these effects were dependent on intact mesencephalic connections, they suggested that amphetamine acted on receptors in the brain-stem reticular formation. This perspicacious suggestion was confirmed by the failure of amphetamine to activate the electrocorticogram after destruction of most of the mesencephalon (Killam et al., 1959). Lesions destroying most of the reticular formation also abolished the alerting effects of adrenaline (Rothballer, 1956). The receptors may be uniformly distributed, for partial destruction of the midbrain reticular formation raised the threshold proportionally to the extent of destruction. Whereas amphetamine readily penetrates to the brain, there is difficulty in accepting a direct action of catecholamines on reticular neurons as tritiated adrenaline and noradrenaline enter the brain with difficulty (Weil-Malherbe, 1961). These techniques give the topographical anatomy of the central receptors. The finer anatomy can be visualized by histochemical methods and defined to a certain extent by the iontophoretic application of drugs. The applied substance can interact with various portions of the postsynaptic membrane of the neuron, with presynaptic fibers, and terminals of other neurons synapsing upon the cell, and even with the neuronal body or processes of these cells (Curtis and Koizumi, 1961). Some localization is possible. Thus the cell may be excited by glutamate; depression of glutamate firing by an iontophoretically applied drug suggests it acts on the postsynaptic membrane. IV. Formation of Catecholamines

The main steps in the formation of adrenaline and noradrenaline (tyrosine+dopa+dopamine+noradrenaline+adrenaline) were proposed by Blaschko (1939) and Holtz (1939). I n the last 15 years the sequence has been confirmed and there appear to be minor additional pathways for synthesis (Fig. 1). First, it was shown that the adrenal medulla could convert noradrenaline to adrenaline (Bulbring, 1949; Biilbring and Burn, 1949). The enzyme L-dopa decarboxylase (Holtz et al., 1938), which converts the amino acid L-dopa to dopamine, was found in the adrenal medulla (Langemann, 1951; Holtz and Bachmann, 1952; Westermann, 1957), as were small amounts of dopamine (Goodall, 1951; Shepherd and West, 1953; Dengler, 1957). Then, Demis et al. (1955, 1956) incubated

r

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00 0

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,

HO /

Tyrosine /

/

/

'

\

Tyramine

\

' \

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\

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Epinine

Adrenaline

Oc topfmine

Synephrine

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N-Methyladrenaline

FIG.1. Pathways in the formation of catecholamines. Solid arrows show the main routes. Enzymes involved are (1) dopa decarboxylase ; (2) dopamine-p-oxide : (3) phenylethanolamine-N-methyltransferase ; (4) catechol-forming enzyme ; (5) nonspecific AV-methyltransferase. (Sdapted from Axelrod, 1963.)

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labeled dopa with hoinogenates of bovine adrenal medulla and identified small amounts of labeled dopamine and noradrenaline. The entire sequence was then established, for in wivo and in witro medullary tissue converts labeled L-tyrosine or L-dopa to labeled dopamine, noradrenaline, and adrenaline (Hagen, 1956; Kirshner and Goodall, 1956; Leeper and Udenfriend, 1956; Masuoka e t al., 1956; Udenfriend and Wyngaarden, 1956; Pellerin and D’Iorio, 1957). The granules in medullary cells are the site of noradrenaline synthesis from dopamine (Kirshner, 1959), whereas decarboxylation of L-dopa to dopamine (Blaschko e t al., 1955) and methylation of noradrenaline to adrenaline take placc in the cytoplasm (Kirshner and Goodall, 1957). There is a similar distribution of ainines and enzymes in sympathetic nerves. Dopamine is found in sympathetic nerves and ganglia (Schumann, 1956; Euler, 1958) ; although in the adrenal gland it constitutes 270, in the central nervous system it amounts to nearly 50% of the total aminc. The distribution of the amine differs in different parts of the brain. The nuclei of the corpus striatum contain mainly dopamine, whereas the hypothalamus and brain stem contain noradrenaline (Bertler and Rosenberg, 1959a,b). Only traces of dopamine are present in the brain stern where it probably functions as a transient precursor of noradrenaline (Carlsson, 1959). The question whether adrenaline can be synthesized in sympathetic fibers is not yet elucidated, although Muscholl and Vogt (1958) found a rather high concentration of adrenaline in prevertebral sympathetic ganglia and it is present in the brain (Vogt, 19541. The key enzyme d o p a decarboxylase occurs in sympathetic nerves and in different parts of the brain (Holtz and Westermann, 1956). The nerve cytoplasm contains L-dopa decarboxylase, dopamine, and noradrenaline (Schumann, 1958) whereas the granules contain noradrendine (Euler and Hillarp, 1956; Euler, 1958). The granules synthesize noradrenaline from dopamine (Goodall and Kirshner, 1958). If labeled L-tyrosine or L-dopa is incubated with homogenates of sympathetic nerves and ganglia, dopamine and noradrenaline are formed (Goodall and Kirshner, 1958) but synthesis does not proceed further. Each step in the formation of catecholainines must be catalyzed by enzymes. Most of these enzymes have been described, but although Kaufman (1959) has studied the conversion of phenylalanine to tyrosine, little is known about the hydroxylation of tyrosine to dopa. The enzymes involved are relatively nonspecific. Dopa decarboxylasc, present in the granules, can decarboxylate tyramine as well as other amino acids (Lovenberg et al., 1962) ; dopamine-p-oxidase not only oxidizes dopamine to noradrenaline but can form octopamine (p-hydroxyphenylethanolamine) from tyramine and adrenaline from Epinine (Bridgers and Kauf-

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man, 1962) ; phenylethanolamine-N-methyltransferase N-methylates octopamine, noradrenaline, and a wide variety of phenylethanolamine derivatives (Axelrod, 1962a) ; a nonspecific N-methyltransferase can N methylate dopamine to Epinine (Axelrod, 1962b). Recently monophenolic amines such as p - and m-tyramine, octopamine, and synephrine have been found to be excreted in the urine (Dewhurst, 1961; Kakimoto and Armstrong, 1962; Pisano et al., 1961). The administration of t y r a ~ i n e - Cand ~ ~ octopamine-C'* resulted in the excretion of trace amounts of noradrenaline and larger amounts of normetanephrine and metanephrine (Creveling et al., 1962). There may therefore be alternative pathways for the formation of catecholamines, and monophenolic amines may serve as precursors for catecholamines (Fig. 1). Axelrod (1963) described the enzymic formation in rabbit liver of adrenaline from synephrine and phenylephrine and dopamine from p - and ml-tyramine. The enzyme was nonspecific and could form catecholamines from substances as diverse as p - and m-octopamine, p-hydroxyephedrine, phenol, stilbestrol, N-acetyl-p-amino-phenol, estradiol, and N-acetylserotonin. The enzyme which hydroxylates L-tyrosine to L-dopa must have a special and selective function for there appears to be no biochemical distinction between L-dopa decarboxylase and 5-hydroxyL-tryptophan decarboxylase. It is thought that the same enzyme serves to decarboxylate L-dopa to dopamine and 5-hydroxy-~-tryptophan (5-HTP) to 5-hydroxytryptamine (Holtz and Westermann, 1957; Westermann et al., 1958; Bertler and Rosengren, 195913). The selection of the correct substrate must take place at the stage before decarboxylation. Tyrosine and tryptophan are present in human serum, but not dopa and 5-hydroxytryptamine phosphate (Jirgl, 1957), so that the blood provides the organs with amino acids which have no direct influence on the distribution pattern of dopamine and 5-hydroxytryptamine (5-HT) . The specificity of amine distribution will presumably be dependent on the ability of different organs to form either dopa from tyrosine or 5-HTP from tryptophan. Cells which store catecholamines may have an enzyme which converts L-tyrosine to L-dopa, whereas cells which store 5-HT have an enzyme which converts L-tryptophan to 5-HTP. In summary, the cells which synthesize catecholamines receive Ltyrosine from the blood and convert i t to L-dopa. This is changed to dopamine by L-dopa decarboxylase in the cytoplasm and stored in granules or converted to noradrenaline by dopamine-/3-oxidase. The noradrenaline may be stored in granules, or in some cells changed to adrenaline by a methylating enzyme in the cytoplasm. I n these cells, the adrenaline must return to the granules for storage.

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V. Preganglionic Nerves and Sympathin Secretion: Adrenal Gland

The adrenal gland is the major source of adrenaline in the body; adrenaline and noradrenaline are liberated on excitation of its preganglionic innervation. There are excellent. accounts of the anatomy (Bourne, 1949), comparative pharmacology (West, 1955), and physiology (Hartman and Brownell, 1949) of the adrenal gland.

A. SYMPATHIN-SECRETING CELLS The predominant amine in the fetal adrenal of most species is noradrenaline; noradrenaline alone is found in the fetal glands of man, rabbit, and guinea pig. Dopamine has been subsequently discovered in adult adrenal glands (Goodall, 1951; Shepherd and West, 1953) but i t is not known whether it has been sought in the fetus. The fetal sheep secretes a lower proportion of adrenaline than after birth (Comline and Silver, 1961). Splanchnic nerve excitation has little effect until about the one hundred and twenty-fifth fetal day when it evokes a two- to threefold increase in sympathin, mainly noradrenaline ; about parturition, considerably more amines with a greater proportion of adrenaline are liberated on nerve stimulation. This is probably due to development of the innervation of the medulla, particularly as the effect was abolished by hexamethonium. Augmented adrenaline secretion from the adrenal gland appears then to be intimately connected with the integrity of adrenal gland innervation. It is interesting that adrenal medullary tumors contain proportionately more adrenaline than found in normal glands a t post-mortem (Holton, 1949). The difference may be due to lack of differentiation of tumor cells, comparable with embryonic rather than adult tissue. The proportion of adrenaline in the gland increases after birth although the distribution of adrenal catecholamines varies in adult vertebrates. In most rodents, noradrenaline is present in small amounts only; in carnivores, it is usually plentiful; in whales, it far exceeds the adrenaline (West, 1955). The amine ratio of the adult cat adrenal gland is extremely variable ranging from 13.3-90.7% noradrenaline (Butterworth and Mann, 1957a). Secretory cells for adrenaline and noradrenaline are found in the mammalian adrenal medulla (for references see Eranko, 1961), in the hen (Eranko, 1957), and lizard (Wright and Jones, 1955). Innervation of the two cell species may be different as indicated by the more intense cholinesterase activity of the noradrenaline- than of the adrenaline-containing cells in the rat, the mouse, and hamster adrenal medullae (Eranko, 1959, 1961) ; one type of cell may lack the methylating system respon-

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sible for converting noradrenaline to adrenaline. The pattern of the noradrenaline cells varies from onc species to another, but in most animals they form clearly delineated islets ; they may be peripherally situated in the medulla as, for example, in the hamster, or randomly distributed. Although there is much vai-iation between species, thc distribution patterns are remarkably similar in the same species. Thc two types of cell survive transplantation (Eriinko, 1956; Coupland, 1958). Othcr observations suggest two independcnt types of cell for sccretion of the adrenal medullary hormones. Insulin depletes medullary adrenaline but not noradrenaline (Eranko, 1952, 1954a; Hillarp and Hokfelt, 1954; Vogt, 1945; Coupland, 1958) ; sinall doses of reserpine cause selective noradrenaline loss (Eranko and Hopsu, 1958; Camanni and Molinatti, 1958). Nicotine produces selective hyperplasia of the noradrenaline-containing cell islets in the r a t (Eranko, 1954b) ; in the mouse, thiouracil increases the size and hormone content of the noradrenaline cell islets (Hopsu, 1960). Although these conclusions dcpend upon histochemical methods, therc is physiological evidence that different types of nerve stimuli may cause preferential secretion of adrenaline or noradrenaline from the adrenal medulla.

B. NERVE SUPPLI A study of the extrinsic nerve supply to the adrenal gland is more than an anatomical exercise for it sheds considerable light on the gland’s function. The adrenal medulla of the cat receives its main nerve supply from the greater and lesser splanchnic nerves. Fihers from the lumbar ganglia of the abdominal sympathetic chain also run directly, or through the celiac ganglia, to the adrenal glands (Elliott, 1913). Stimulation of branches from the lumbar ganglia liberate sympathin (Cannon e t al., 1926). According to Maycock and Heslop (1939), additional secretory fibers derive from the first and second and occasionally the third lumbar sympathetic ganglia. The innervation is probably more extensive (Marlcy and Prout, 1964); in general, excitation of the nerves rising from the last thoracic and upper three or four lumhar sympathctic ganglia and which pass to the celiac and aorticorenal ganglia will liberate adrenal sympathin. The final shaping of adrenal medullary secretion depends upon the relation of the nerves within the medulla to the secretory cells. The exact mode of nerve termination in the medullary cells is disputed. According to Hollinshead (1936), as the nerves reach the medulla they often branch immediately, breaking up into smaller trunks, but sometimes they can be traced practically intact some distance. The various smaller nerves

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branch and rejoin each other and neighboring trunks in a bewildering fashion, eventually forming a delicate plexus in the connective tissue around the medullary cell groups, with still more delicate fibrils present between the cells of a group. Although the smaller nerve fibers in the medulla branch and cross each other frequently, no clear anastomoses have been observed. Hillarp (1946) examined the innervation of various autonomic organs. A nervous ground plexus occurred in the adrenal medulla; each cell was in direct contact with some part of the ground plexus. Within the plexus each axon innervated a number of cells, the secretor unit. As several neurons terminate in the secretor unit, the response to indirect stimulation could be modified by temporal and spatial summation. The terminal plexus would not be a continuous network as understood by Stohr (1935), which requires that nervous excitation would activate all parts of the medulla.

C. RESTINGSECRETION OF AMINES Early experiments on adrenal gland denervation (Stewart and Rogoff, 1916, 1917) indicate that secretion falls to undetectable levels; a constant small secretion of catecholamines from acutely or chronically denervated adrenal glands was subsequently found (Vogt, 1952; Duner, 1953; Rapela, 1956). I n the cat, after chronic denervation of both adrenal glands, the mean secretion per minute is 21 ng adrenaline and 34 ng noradrenaline (Vogt, 1952). Acute nerve section reduces the output of both amines on an average by 60-70%, sometimes to almost undetectable levels. There is a subsequent rise in output after nerve section, in the absence of nerve stimulation, or of any other stimulus to secretion (Marley and Paton, 1961). Before nerve section the stores of amines in the glands are possibly slightly depleted by the normal activity of the splanchnic nerve ; when this drain on the stores is prevented, amines accumulate until a spontaneous “overspill” occurs. D. DISCHARGE OF ADRENAL SYMPATHIN 1. Drugs Actin*gDirectly on the Adrenal Medulla The first proof that acetylcholine release from preganglionic nerves led to the liberation of adrenergic substances was with the adrenal gland (Feldberg et al., 1934). Earlier, Dale and Laidlaw (1912) had shown liberation of adrenaline from the adrenal gland by pilocarpine. Liberation of sympathin by acetylcholine and by splanchnic nerve excitation, although mainly due to a nicotine type of action and one which is abolished by large doses of nicotine, is also partly due to a muscarinic action of acetylcholine which is abolished by small doses of atropine (Feldberg

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et al., 1934). Arecoline, another parasympathomimetic substance, liberates sympathin because of its nicotine-like action. There is no evidence that adrenal medullary cells depolariae like nerve cells, but acetylcholine in somc way alters the property of the membrane (Hagen, 1961). The effect of acetylcholine may be to cause calcium ions to penetrate the adrenal medulla sccretory cells (Douglas and Rubin, 1961). It seems that the process of medullary secretion involves a complex chain of events (referred to as “vesiculation” and membrane flow) during which the secretory granules are first attached to the cell membrane and then their contents extruded through it (de Robertis et al., 1960) ; this may be the process which is calcium-dependent. Another view is that on nerve excitation the granules, without migrating, spill their amines which diffuse into the clear cytoplasm and are then released (J. D. Lewis and Lever, 1960). Calcium is known to influence not only cell membranes but also the physicochemical properties of cell sap (Heilbrunn, 1956) ; Hodgkin and Katz, 1949; Chambers and Kao, 1952). A number of other choline esters, including carbamylcholine and benzoylcholine, provoke adrenal sympathin dischargc without prcfercntial adrcnalinc or noradrenaline secretion (Butterworth and Mann, 1958) ; acetyl-P-methylcholine is ineffective. Possibly the choline esters act on the membrane of the chromaffin cell causing an increase in membrane permeability (Eade, 1957) ; there is no direct action of the choline esters on the storage granules. Other basic substances such as 5-hydroxytryptamine (Reid and Rand, 1952), coniine (MacFarland, 1944), and histamine liberate sympathin. Adrenal sympathin is released by ferrous (Eicholtz and Roesch, 1949), bile (Emmclin and Muren, 1949), and potassium salts (Houssay and Molinclli, 1925; Vogt, 1952). 2. Drugs Acting Indirectly on the Adrenal Gland

Sympathin may be liberated from thc adrenal medulla by morphine, tetrahydronaphthylamine, and the gaseous anesthetics acting on the central nervous system (Elliott, 1912). Vogt (1954) clegantly clarified the relation between central and peripheral sympathin release; this will be considered later. Increased sympathin secretion is evoked by injecting lactic acid, sodium carbonate, ammonium chloride, procaine, decaborane, and barium or calcium chloride into the aorta (Woods et al., 1956). Whether this is due to a direct or an indirect action is not known. 3. Drug Effects after Partiat Nerve Section

Such studies are important for the light they shed on the innervation and activation of adrenal medullary cell complexes. I n rats killed a t the height of insulin hypoglycemia the histology of the adrenal medulla

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departs considerably from normal. Apart from a few cell complexes with strong chromaffin staining, the rest are depleted of chromaffin substance. The cell cytoplasm is shrunk and contains large vacuoles; the nuclei are pyknotic (Hillarp, 1946). However, after partial unilateral denervation of the adrenal gland, insulin hypoglycemia leaves many cells unaffected ; others are submaximally activated, whereas loss of chromaffin substance is found in few cell complexes. From these and other experiments, Hillarp (1946) suggested that the adrenal medulla is organized into functional units of cell complexes which may be activated more or less independently of each other.

E. SPLANCHNIC NERVE EXCITATION The ideal in studies of adrenal medullary function is to separate the adrenaline and noradrenaline secreted before assay, as in the experiments of Vogt (1952). This is time-consuming and instead the mixture of amincs is often tested on two discriminating test objects. The electrically stimulated rat uterus (Harvey and Pennefather, 1962) is a t least 10,000 times less sensitive to noradrenaline than to adrenaline, so it may be assumed to respond only to adrenaline in the sympathin released from the adrenal gland. Unfortunately a satisfactory assay tissue discriminating in favor of noradrenaline is not available; the pithed r a t blood pressure method is only 2-5 times more sensitive to noradrenaline than to adrenaline. If the amines are not separated before assay, i t is important to try and confirm the results obtained with in vitro by in vivo assay. I n vivo assay gives an idea of the time course of sympathin secretion. 1. Threshold Frequency of Excitation for Sympathin Release

This is determined both by the intensity of and frequency of excitation. I n the dog the threshold intensity for sympathin secretion on excitation of the major splanchnic nerve was 4.5 k 0.8 v (Mirkin, 1961). The threshold intensity probably depends on excitation rate, faster excitation disposing to temporal summation. In the cat, excitation a t 8-64/second with 2 v but not 1 v elicited sympathin discharge. However, supramaximal excitation a t l/second to the major splanchnic nerve was apparently ineffective in liberating sympathin unless sustained for 15 minutes; consistent release was obtained with 24/second excitation (Marley and Paton, 1961). With the in vivo assay techniques secretion could have gone undetected because of binding of sympathin by tissues between the adrenal gland and the assay tissue. There is probably rapid uptake of sympathin in the gland’s vicinity with exponential decline of circulating sympathin corresponding to the distance from the adrenal gland, By monitoring output direct from the gland with a modification of Vane’s (1958) blood-bathed isolated organ tech-

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nique (Marley, 1961a) increased secretion was detected with 2 supramaximal l/second shocks to the major splanchnic nerve and 5 shocks to the smaller nerves (Fig. 2A). The shocks were 100 msec apart, an interval too long for acetylcholine to persist a t the preganglionic terminals. One could postulate an excitatory state, set up in the gland cell by the transmitter, intense enough to initiate discharge of amines. By two different methods of determination the quantity secreted per shock a t l/second excitation was 5-10 ng sympathin (Marley and Prout, 1964). Once secreted, the sympathin may be taken up by receptors within the adrenal gland, similar to uptake of noradrenaline in the spleen described hy G. L. Brown and Gillespie (1957). The low threshold of sympathin secretion militates against the importance of uptake in the adrenal gland. Reabsorption of amine by the sccretor cell may also occur (Paton, 1961a). Paton suggests that in nerve endings and in the adrenal medulla the sympathin is the dominant intracellular cation, like potassium in many other cells, released when the membrane potential is reduced, and sucked back, recovered, and returned to store when the events of excitation are over. 2. Excitation Rate for Optimal Sympathin Secretion

To compare secretion with different rates of excitation to the splanchnic nerves the number of shocks applied should be constant. This principle was used elegantly by G. L. Brown and Gillespie (1957) in investigating output of sympathetic transmitter from the cat’s spleen. The optimal frequency of excitation for maximum response of the majority of autonomic receptors is 20-30/second (Cannon and Rosenblueth, 1937) but this may not be applicable to the splanchnic medullary cell synapse. Certain of the autonomic receptors, e.g., the cat’s denervated nictitating membrane, have been used for estimating circulating sympathin. It is quite likely that they respond maximally to submaximal sympathin secretion and so are unsuitable for measuring optimum output of the adrenal gland. This criticism does not apply to in vitro assay of adrenal sympathin nor to in vivo assay with tissues suspended in an extracorporeal circulation (Vane, 1958) which can be made as sensitive as required so as to give graded responses over the appropriate range. With bursts of 60-180 stimuli to the major splanchnic nerve (Fig. 2B, C) optimal secretion from the adrenal medulla occurred with 30-60/ second excitation. Brief bursts of stimuli were given as secretion declined with protracted excitation (Fig. 2D, E) . With 200 stimuli or less the output per stimulus was about 3-5 ng sympathin (Marley and Prout, 1964). While the excitation rate for optimal sympathin secretion was constant

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for any one cat there was a good deal of variation around the optimum response in different cats. In other species, optimal sympathin secretion was elicited with excitation a t 15-40/second (Mirkin, 1961; Rapela, 1956; Silver, 1960) ; maximal secretion in the dog was occasionally found with 160/second excitation (Rapela, 1956). Excitation for optimal secretion was slower for the smaller splanchnic nerves, about 16/second for the smallest nerve and between 16-30/second for the intermediate-sized nerves (Marley and Prout, 1963). If the smaller nerves are composed mainly of C fibers of small diameter they would be liable to prejunctional failure and this imposes a, lower limit for optimal excitation. Even in the frog’s skeletal muscle-nerve preparation prejunctional failure occurs with stimulation much above 10/second (Krnjevi6 and Miledi, 1958, 1959). Difference in the number of secretor units innervated by the smaller nerves may also contribute to the differences in optimal excitation rates. Secretion was greater on exciting the larger than the smaller splanchnic nerves. Sympathin output dwindled with excitation faster than the optimum, even if the number of stimuli was increased. Decline in secretion with faster excitation has been attributed to successive stimuli falling within the refractory period (Rosenblueth, 1932). The refractory period of the adrenal ncuroglandular synapse has been assumed to be the same as that in the cervical sympathetic ganglion, 20-30 msec (Bishop and Heinbecker, 1930). The theoretical upper frequency response of the synapse would be 30-50/second, which corresponds to the range of optimal response for sympathin secretion. Temporal dispersion of impulses may be a limiting factor, a t least in the greater splanchnic nerve; conduction velocities vary from 75 m/sccond for constituent A fibers to 1 m/second in the C fibers (McLeod, 1958). Certain cells cease to respond with excitation faster than 40/second in the cat’s stellate ganglion and this was ascribed partly to impulse dispersion (Larrabee and Bronk, 1947). As each supramaximal impulse theoretically liberates the same amount of acetylcholine a t the neuroglandular synapse, the increase up to the optimal secretion with faster excitation has been attributed to temporal summation (Celender, 1954). Young (1939) showed that the adrenal medulla was divided into functional units and suggested they could be separately recruited by activation of the neurons. 3. Spatial and Temporal Summation in the Adrenal G1an.d Spatial summation can be tested by exciting the splanchnic nerves with varying intensity and constant number of shocks a t constant fre-

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quency; temporal summation can be tested by varying excitation rate with constant number of shocks and intensity. At slower excitation (8,16/second) secretion rose linearly with increase in stimulation intensity to the greater splanchnic nerve, to a maximum at about 10 v. With faster excitation (32,64/second) output was virtually maximal a t 3 v (Marley and Prout, 1964). At slow rates of firing in the splanchnic nerve spatial is therefore a t least as important as temporal summation. The slow excitation frequencies correspond to the rates of physiological discharge in postganglionic sympathetic nerves (Folkow, 1952). With faster excitation, which may correspond to the discharge frequency in emergency conditions such as asphyxia or hemorrhage, temporal summation took precedence. As supramaximal stimuli were given in the experiments for the optimal rate of excitation of the splanchnic nerves, increase in secretion with faster excitation would be due to temporal summation. Spatial summation is a quantitative phenomenon ; temporal summation may depend on qualitative and quantitative factors. After partial section of the splanchnic nerve, to test the importance of the number of neurosecretor units innervated, the optimal excitation rate for secretion was sometimes changed from 60 to 30/second; output was reduced FIG.2. Sympathin liberation on splanchnic nerve excitation. A, C, and D. In vivo assay of sympathin using extracorporeal circulations. R a t stomach strip mperfused by carotid arterial and adrenal venous blood (A) and carotid arterial blood only (C, D). B and E. In vitro assay of adrenaline and noradrenaline with the isolated rat uterus and pithed rat blood pressure methods, respectively. A. Threshold. Chloralosed cat, 2.4 kg. On left, relaxation of stomach strip with 50 ng adrenaline injected into the adrenal extracorporeal circulation; on right, relaxation of stomach strip on excitation (S) with 6 shocks a t l/second to the left major Rplanchnic nerve. €3 and C. Optimal sympathin secretion. B. Chloralosed cat, 3.0 kg. Excitation with 180 stimuli of left major splanchnic nerve. Excitation frequencies shown at arrows; greater adrenaline and noradrenaline secretion with 32/second than with 1, 4, or 16/second excitation. C. Chloralosed cat, 3.4 kg. On left, relaxation of stomach strip with adrenaline (1.0 pg, iv) injected into cat. On right, relaxation of stomach strip on excitation (S) with 120 or 240 shocks to the right major splanchnic nerve. Optimal response with 3264/second excitation. D and E. Fatigue on sustained, or splanchnic nerve excitation with inadequate rest intervals. D. Chloralosed cat, 4.0 kg. On left, relaxation of stomach strip with adrenaline (0.5 pg, iv) injected into the cat. On right, sympathin output dwindles (return of tone in rat stomach strip) on sustained 32/second excitation (S)of left major splanchnic nerve. E. Chloralosed cat, 2.4 kg. Adrenaline and noradrenaline secretion before and after section of the left major splanchnic nerve (downward pointing arrow) and on excitation of the left major splanchnic nerve. Solid bars give duration of excitation. Optimal response at S/second excitation with decline on 16 and 32/second excitation because of inadequate rest intervals between stimulations.

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most in the excitation range in which temporal summation is most effective. With almost complete section secretion on nerve excitation was drastically reduced and virtually unaltered over 16-64,kecond excitation (Marley and Prout, 1964). Temporal summation is therefore more effective the larger the number of neurosecretor units activated. 4. Splanchnic Nerve “Fatigue)’ Elliott (1912, 1913) found that after prolonged stimulation of the splanchnic nervc the effectiveness of excitation was reduced, although adrenal sympathin was not depleted. Unresponsiveness of the adrenal gland after prolonged nerve excitation may be due to prcsynaptic, synaptic, or postsynaptic failure. The average content of amine in the adrenal gland is 320 pg (Butterworth and Mann, 1957a)) and if with optimal excitation about 5 pg/ minute is secreted, then 1-276 of the medullary contents would be released per minute. Exhaustion of aniine should occur within 50-100 minutes unless resynthesis or re-storage takes place. However, secretion declines exponentially over 15-45 minutes (Fig. 2 D ) . Postsynaptic failure (depletion of amines) was unlikely as nicotine or histamine still evoke vigorous sympathin discharge. This argument is not crucial if there is a quanta1 arrangement of the nerve supply, for nicotine or histamine will liberate sympathin from an unexcited portion of the gland innervated by other nerves. The rapid recovery is a stronger argument for a prcsynaptic or synaptic fatigue; even after prolonged nerve stimulation, a rest of 5-15 minutes is sufficient to restore the response to nerve excitation (Marley and Paton, 1961). I n contrast, after depletion of adrcnal sympathin with acetylcholine there was no replacement within 15 hours, and restoration of amine required 6-7 days (Butterworth and Mann, 1957b). It has been suggested that amine synthesis is so fast that no depletion occurs on splanchnic nerve excitation (Holland and Schumann, 1956). Eade and Wood (1958) challenged this observation and concluded that not only was amine synthesis not increased during splanchnic nerve excitation but depletion was probable. The rate of onset of fatigue depended on the rate of splanchnic nerve excitation, developing more rapidly with fast excitation (Marley and Paton, 1961). Although recovery was rapid from fatigue, if rest intervals between excitation periods were inadequate secretion dwindled (Fig. 2E). Adrenal sympathin secretion on nerve excitation is mediated through acctylcholine release a t preganglionic endings in the medulla. With prolonged excitation over a wide range of frequencies the acetylcholine out-

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put of a sympathetic ganglion falls exponentially to a final constant rate per minute. This implies that a single stimulus released less acetylcholine with fast than with slow excitation (Perry, 1953). The secretion of the adrenal medulla declines in parallel fashion on sustained excitation. With 10 or less shocks to the major splanchnic nerve, secretion was 5-10 ng per shock; if acetylcholine was allowed to accumulate after eserine, the secretion per shock rose fourfold. With faster excitation but with 200 shocks or less, approximately 3-5 ng sympathin per stimulus was obtained. If excitation was continued for 8 minutes, output declined to 1 ng per shock, and with sustained excitation output was 0.25 ng sympathin or less for a single stimulus (Marley and Prout, 1964). This would suggest that fatigue on exciting the splanchnic nerve with optimal or slower excitation was a type of synaptic failure due to decrease in the amount of transmitter liberated. With faster than optimal excitation, fatigue could be also due to prejunctional failure. Fatigue was easier to elicit with the smaller than with the larger splanchnic nerves. The difference in susceptibility to fatigue (with optimal or slower excitation rates) was related quantitatively to the number of secretor units innervated, for the gland’s response to stimulation of the smaller nerves could be simulated by exciting the partly cut major splanchnic nerve (Marley and Prout, 1963). There may be a change in the amine ratio during prolonged nerve excitation. In 5 of 9 experiments with intermittent splanchnic nerve excitation, the adrenaline proportion fell (Bulbring and Burn, 1949). Evidence for a more rapid decline of adrenaline than of noradrenaline secretion during sustained excitation of the splanchnic nerve was obtained with in vivo (Marley and Paton, 1961) but not by in vitro assay (Marley and Prout, 1964) ; that adrenaline secretion may be tenuous was evident from the usually greater recovery of noradrenaline secretion on re-excitation with a rest interval after fatigue. It is worth noting that, in any case, the amine ratio of the cat’s adrenal gland is extremely variable, ranging from 13.3-90.7% noradrenaline (Butterworth and Mann, 1957a). 5. Blood Sympathin Content and Adrenal Medullary Secretion

Sympathin liberated by the adrenal gland affects its resting secretion and the response to splanchnic nerve excitation. This is not surprising for adrenaline has a depressant action on sympathetic ganglia (Marrazzi, 1939; Matthews, 1956); facilitation as well as depression may occur (Marrazzi and Marrazzi, 1947). The amount of adrenaline liberated from the dog’s perfused adrenal gland on splanchnic nerve excitation depends on the concentration of adrenaline in the perfusing blood (Bulbring et al., 1948). With little

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adrenaline in the perfusate, splanchnic nerve excitation discharges only small amounts of adrenaline; with increased adrenaline content, the discharge on excitation rose steeply. If the adrenaline was increased beyond a certain optimum, secretion declined and was finally abolished. With cross-perfusion experiments MalmCjac (1955) showed that small doses of adrenaline (O.Frl.0 pg/kg infused/minute) enhanced secretion ; with large amounts (12-15 pg/kg/minute) secretion dwindled.

6. Denervation of the Adrenal Medulla

If the presynaptic nerves to the adrenal gland are cut, the chromaffin medullary cells do not degenerate. Three to 6 weeks after partial denervation of the adrenal medulla, excitation of the remaining intact nerves liberated 2-8 times more sympathin than from the normal control gland (Simeone, 1938). The adrenal gland is homologous with sympathetic ganglia which are sensitized to acetylcholine and nerve impulses on partial denervation. That the gland is sensitized by partial denervation supports the idea of independent innervation of areas within the adrenal medulla. The nerves to the adrenal gland regenerate 3 months after denervation (Hollinshead and Finkelstein, 1937). 7. Crossed Innervation of Adrenal Gland

It has been held that there is no crossed innervation to the cat’s adrenal medulla (Cannon et al., 1926; Maycock and Heslop, 1939). However, in acute denervation experiments sympathin discharge ostensibly from the left adrenal gland was elicited on exciting the right major splanchnic nerve (Marley, 1961a). Whereas 2 shocks a t l/second excitation to the ipsilateral nerve was sufficient to liberate sympathin, a t least 100 shocks a t 10/second was required on contralateral nerve excitation. Output was abolished by dividing the leash of fibers connecting the aorticorenal ganglia above and below the origin of the superior mesenteric artery. Secretion from the adrenal medulla was a t least 10 times greater on exciting the direct than the crossed fibers (Marley and Prout, 1964) ; fatigue developed with sustained stimulation. 8. Sensitivity to Ganglim-Blocking Agents This is similar to that of autonomic ganglia. Nicotine excites and then paralyzes. The sensitivity to methonium compound of different chain lengths C, to C, was similar to that of ganglia; C, was the most potent and decamethomium (Clo) ineffective. As with the ganglionic synapse, the splanchnic-suprarenal synapse was more readily blocked

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when continuously activated than when briefly and intermittently stimulated (Marley and Paton, 1961). Selective block of the adrenal medulla, preventing secretion of sympathin but without affecting other ganglia, is claimed with the substance N,N-diisopropyl-N’-isoamylN’-diethylaminoethyl urea (Gardier et al., 1960). Atropine acts weakly a t the synapse (Feldberg et al., 1934).

F. EXTIRPATION OF THE ADRENAL GLANDS Increase in noradrenaline but a great reduction of adrenaline excreted in the urine occurs after adrenalectomy in man (Euler et al., 1954). The disappearance after bilateral adrenalectomy of adrenaline from the peripheral plasma suggests its origin from the adrenal medulla and that the plasma adrenaline concentration in the intact subject is an index of the gland’s activity (Munro and Robinson, 1960). The plasma noradrenaline rises after adrenalectomy; this suggested to Munro and Robinson (1960) that noradrenaline is unlikely to form any considerable part of medullary secretion in man. There is doubt whether, in the intact animal, any of the noradrenaline secreted a t postganglionic adrenergic nerve endings finds its way into the circulation (Celender, 1954). With extracorporeal circulation experiments in the cat, the resting plasma noradrenaline and, to a lesser extent, adrenaline values were raised compared to values in similarly prepared animals but without extracorporeal circuits (Marley and Prout, 1964). When normal human subjects change from the supine to the sitting position the plasma noradrenaline rises (Munro and Robinson, 1960). These findings suggest increased activity of the vasoconstrictor nerves associated with an escape of noradrenaline from the endings into the peripheral blood.

G. CENTRALCONTROL OF ADRENAL MEDULLARY SECRETION Central nervous function is investigated classically by ablation, transection, or electrical stimulation. Such methods shed light on the relation of the central nervous system to the adrenal medulla. Adrenal medullary secretion is influenced by a center in the medulla oblongata subject to reflex inhibition or excitation (Elliott, 1912 ; Cannon and Rapport, 1921; Tournade and Malmhjac, 1932). Stewart and Rogoff (1920) felt rather that the adrenal glands were controlled from the cervical spinal cord, as transection between the last cervical and the fourth thoracic segments abolished spontaneous sympathin secretion; the center was bilaterally represented, for cord hemisection suppressed ipsilateral secretion without affecting that from the contralateral gland. Brooks (1933) dissented on the grounds that spinal cord mechanisms modifying adrenal secretion would be reflex in nature. He confirmed that

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afferent stimuli evoked adrenal sympathin secretion after the brain had been disconnected from the spinal sympathetic outflow. An area inhibiting adrenal medullary secretion has been located in the cat between the optic chiasma and the superior colliculi (Rogoff e t al., 1946). Its identification was based on experiments in which increase in spontaneous sympathin secretion followed transection between the superior colliculi and thc optic chiasma but not after anterior transections. However, higher nervous influences nornially act tonically on adrenal medullary secretion; plasma adrenaline was significantly lower in human subjects with transverse spinal cord lesions above the fourth thoracic segment, disconnecting the main outflow of secretory fibers to the adrenal medulla, than below (Munro and Robinson, 1960). With lesions below the sixth thoracic segment, plasma adrenaline concentration was normal. These conflicting findings are not necessarily irreconcilable for they may be due to the presence of inhibitory and excitatory centers in the brain stem. According to Ingram (1960) autonomic mechanisms in the spinal cord are subject to facilitatory and inhibitory influences from higher centers. Identification of such centers and connections has gone hand-inhand with the refinement of electrophysiological techniques. Liberation of sympathin occurs on hypothalamic excitation (Houssay and Molinelli, 1925; Magoun et al., 1937). Adrenal sympathin secretion is enhanced by exciting the brain-stem reticular formation in the cat (Marley, 1 9 6 1 ~ ) .The sympathin liberated was monitored with Vane’s (1958) blood-bathed isolated organ technique ; secretion could not bc elicited after section of the spinal cord a t C.l and was virtually abolished by bilateral splanchnic nerve section if the spinal cord was intact. A greatly enhanced proportion of adrenaline in the adrenal medullary secretion followed excitation of the hypothalamus in the cat (Briicke et al., 1952). The composition of the sympathin secreted was determined by the location of the stimulus. Excitation in the upper posterior paravcntricular areas generally give rise to high adrenaline and low noradrenalinc proportions but the reverse for stimulation of adjacent areas (Folkow and Euler, 1954). The adrenal gland and other abdominal viscera have projections over afferent pathways (Downman, 1955 ; Downman and Evans, 1957) to higher centers (McLeod, 1958) permitting modulating feedback influences.

H. ADRENALMEDULLARY SECRETION AND THE CENTRAL NERVOUSSYSTEM : AWARENESS The relation of adrenal sympathin to awareness will be considered briefly. To study the effect of adrenaline on the reticular-activating

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system, Dell (1961) used cats with brain-stem section at the intercollicular level and in the middle or anterior third of the pons. The neocortical electrical activity of such a preparation is of the sleep pattern. Adrenal sympathin secretion evoked either by excitation of the splanchnic or sciatic nerves produced electrocortical arousal (Bonvallet et al., 1954). Similar results were obtained in cats with coagulation of the posterior portion of the reticular formation (Rothballer, 1956). A “neurohumoral” factor may be liberated into the blood stream on excitation of the brain-stem reticular formation (Purpura, 1956). In cross-circulation experiments, electrical excitation of the brain stem in the donor cat produced, after a 30-80 second delay, electrocortical activation in the recipient cat; in the animals with transection of the spinal cord a t C.l, the substance could not have originated from the adrenal medulla. The substance was unlikely to be adrenaline or noradrenaline for in the extracorporeal circulation experiments as little as 50-100 ng adrenaline could be detected; none appeared in the circulation on excitation of the reticular formation once the spinal cord had been sectioned (Marley, 1 9 6 1 ~ )Smaller . quantities of sympathin would have been ineffective as adrenaline (1-2 p g / k g intravenously) is required in the adult cat encipphale isole’ preparation for electrocortical arousal. The adrenal cortex is more intimately involved than the medulla in central nervous activity; adrenocortical insuffiency is associated with susceptibility to central depressant drugs. S. Cook e t al. (1960) gave pentobarbitone to cats in which bilateral adrenalectomy had been done 4-5 weeks previously. Electrocortical activity during electrical stimulation of the brain-stem formation was recorded; the end point was that a t which brain-stem stimulation failed to elicit electrocortical arousal. Adrenalectomized cats maintained on deoxycorticosterone were more susceptible to pentobarbitone than those supported by cortisone, whereas those with both steroids replaced were no more susceptible than normal animals. It was construed that adrenal medullary secretion played no significant role in counteracting the central depressant effects of pentobarbitone. Contradictory results were obtained from experiments in which disturbance of adrenocortical function was avoided; there was increased sensitivity to the central depressant alcohol, methylpentynol, after chronic denervation of the adrenal gland in cats (Adams e t al., 1962). Methylpentynol was infused until electrocortical arousal could not be obtained and the drug concentration in the brain determined. The methylpentynol concentration was significantly higher in normal animals and those with partial adrenal denervation than in those with bilateral adrenal denervation.

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VI. Postganglionic Sympathetic Nerves and Sympathin Secretion: The Spleen

It is instructive to compare the effect of nerve excitation on sympathin release from the spleen with that from the adrenal medulla. At first sight the two situations are very different. Whereas the innervation of the spleen is postganglionic, that of the adrenal gland is preganglionic. I n the spleen, the sympathin released traverses a considerable bulk of tissue before appearing in the venous blood and uptake of amine occurs in the tissue; in the adrenal gland, sympathin can be collected before reaching its receptors. Splenic nerve excitation releases mainly noradrenaline (Peart, 1949; Mann and West, 1950), the adrenaline content being seldom more than 10% (Mirkin and Bonnycastle, 1954) ; variable proportions of adrenaline and noradrenaline are secreted from the adrenal medulla. The following account is taken from the exemplary papers of Brown and his colleagues (G. L. Brown, 1961; G. L. Brown and Gillespie, 1957; G. L. Brown e t al., 1959, 1961). The amount of sympathetic transmitter overflowing into the splenic venous blood on splenic nerve excitation depends on a balance between the amount liberated and the proportion taken up on receptors, the two phenomena being variable. A. LIBERATION OF TRANSMITTER Liberation of transmitter is determined by two factors. These include (1) frequency of nerve excitation: With 200 stimuli a t excitation slower than lO/second sympathin could not be detected in the splenic venous blood. As the excitation frequency increased from 10 to 30/second the output of noradrenaline rose to a maximum, thereafter declining so that a t 300/second little was detectable. The failure in output was probably TABLE I NORADRENALINE LIBERATED (pg/STIMULUS) FROM CAT'S SPLEEN SPLENICNERVES

Condition

No prior section of splanchnic nerve Prior section of splanchnic nerve to decentralize spleen a

b

Excitation frequency/second (200 stimuli) 10 30 10 30

Data from G . L. Brown el al. (1961). Number of observations in parentheses.

Control' 169 841 148 394

(25) (36) (6)

(29)

ON

EXCITATION OF

After adrenergicblocking agents (Dibcnzyline or Hydergine)* 1710 1060 3060 1800

(8) (14) (7) (5)

ADRENERGIC SYSTEM AND SYMPATHOMIMETIC AMINES

199

due to prejunctional failure since the nerve trunks were C fibers of small diameter. The amount of noradrenaline liberated per stimulus is given in Table I. The other determinant (2) is nerve rest: The postganglionic neurons to the spleen can be rested by decentralizing them; overflow of noradrenaline a t 30/second excitation was halved, but paradoxically that a t 10/second was normal (Table I). The decrease in overflow was fully developed 24 hours after nerve section; there was no change in the ratio of the noradrenaline to adrenaline liberated. Overflow a t 30/second stimulation could be restored by exposing the nerve to a train of conditioning stimuli; thus in 5 experiments the mean initial overflow a t 30/ second excitation was 381 pg/stimulus, whereas after a train of lo00 stimuli a t the same frequency the mean overflow was 936 pg/stimulus. The effects of splanchnic nerve section on overflow could be reproduced with ganglion-blocking agents.

B. UPTAKE OF NORADRENALINE ON SPLENIC RECEPTORS This was determined by (1) nerve rest: The diminished overflow a t 30/second excitation after nerve rest could be due to reduced liberation or to increased uptake of noradrenaline. As administration of an adrenergic-blocking agent restored overflow to normal levels (Table I ) the reduced overflow was apparently due to increased amine uptake on splenic receptors. The other determinant was (2) receptor avidity: The curve relating the amount of transmitter in the splenic venous blood t o frequency of nerve excitation was not altered by the administration of monoamine oxidase or O-methyltransferase inhibitors; the amount of noradrenaline liberated was therefore independent of amine destruction. However, a dramatic change was produced by substances such as phenoxybenzamine blocking the effects of injected noradrenaline or of nerve stimulation on the tissue. The amount of transmitter appearing a t lO/second excitation was increased nearly tenfold (Table I) whereas a t 30/second there was a doubtful significant increase of noradrenaline. Output of transmitter a t l/second excitation could now be detected. It was concluded that the splenic nerve endings liberate noradrenaline and that this is taken up a t receptor sites on the splenic muscle cells. Combination with the receptors was a necessary prelude to the destruction and removal of liberated noradrenaline ; the receptors might destroy noradrenaline in a way similar to that suggested by BupanEiE (1953) for acetylcholine. The process of uptake on receptors was slow. At lO/second stimulation, 90% of the liberated transmitter was taken up by the tissue; a t 30Jseeond excitation the rate of liberation was twice that at lO/second but only 28% of the liberated transmitter was removed

200

E. MARLEY

by the tissue. Faster excitation depressed the uptake mechanism, reminiscent of the desensitization of cholinergic receptors by the iontophoretic application of acetylcholine (Katz and Thesleff, 1957) or by repetitive stimulation of the motor nerve (Thesleff, 1959). Once the uptake mechanism was swamped, or the receptive sites blocked by phentolamine, the great bulk of the liberated noradrenaline overflowed. Paton (1961a) referred to the curious similarity of sympathin output on splenic and splanchnic nerve excitation; thus, in both, optimal output occurred a t about 30Jsecond excitation with decline in the amount of amine liberated on faster or slower excitation. Paton suggested that the similarity was due to the capacity of amine-secreting tissue, a t rest or after recovery from excitation, to reabsorb amine. VII. Peripheral Action of Sympathomimetic Amines: Structure-Activity Studies

As little is known about the actual nature of adrenergic receptors there are shortcomings to all structure-activity studies. Our knowledge is limited to the chemical structure and certain physicochemical properties of the drug molecules, leaving no alternative than to make observations relating the structure of a compound with its effects on selected systems (Ariens, 1956). The sympathomimetic amines can be tested on a tissue which they excite, e.g., the dog’s retractor penis, or inhibit, such as the rat uterus; excitatory or inhibitory potency can be compared. The response of smooth muscle t o adrenaline should be regarded as the resultant of two opposing actions, so there are tissues which respond biphasically depending upon conditions. However, a single tissue may respond antipodally in a predictable fashion not just to adrenaline but to a series of structurally different amines. While the methods of testing are complementary, studies with the one tissue are likely t o be more informative. The phenylethylamine series is unusual in that many modifications of structure can be made without destroying activity.

A. No DIFFERENTIATION BETWEEN ADRENERCIC RECEPTOR AND ADRENERGICNEURON The classic work is that of Barger and Dale (1910), who tested a large number of amines for their excitor (pressor) or inhibitory action (relaxation of the virgin cat’s uterus). Many amines possessed adrenaline-like activity, the simplest being the primary fatty amines. The optimum carbon skeleton for sympathomimetic action consisted of a benzene ring with a side chain of two carbon atoms, the amino group and the benzene nucleus each being attached to a different carbon atom of the side chain. Activity was increased by phenolic hydroxyls in the 3,4-position relative to thc side chain; when these were present, the in-

ADRENERGIC SYSTEM AND SYMPATHOMIMETIC AMINES

201

troduction of an alcoholic hydroxyl attached to the carbon atom linked

to the ring increased potency further. Excitor and inhibitor sympathomimetic activity varied to some extent independently. Of the catechol bases those with a terminal NH-CH, group reproduced inhibitory sympathomimetic effects more powerfully than excitor; the opposite was true of the primary amines of the series. Exceptions t o the arrangement of an amino and a phenyl group on adjacent carbon atoms for optimal pressor activity have since come to light. These include the development of 2-aminoheptane, and replacement of the phenyl nucleus by the hydroaromatic cycle or by the a-thienyl nucleus (Hartung, 1945). Knowledge up to 1945 for the effect of substitution in the phenylethylamine molecule is summarized in Table 11. Barger and Dale (1910) suggested that the primary amines were imperfect sympathomimetic substances for they possess little inhibitory action. Lands and Tainter (1953) investigated the structural requirements for inhibitory action (Table 111). The structural needs were as specific for inhibitory as for excitatory effects ; relatively small changes in molecular structure radically modified activity. Lands and Tainter (1953) tested the substances for bronchodilator action (inhibitory) in the guinea pig and pressor (excitatory) effects in dogs. Paradoxically some of the modifications favorable for excitation also disposed to inhibition, but this was possibly a consequence of testing amines on different tissues; the threshold for opposing actions differed in different organs. The pressor is not an ideal test response for i t is a composite reaction in which increased cardiac output and changes in splanchnic, skeletal muscle, and skin blood volumes all contribute. I n general, the most effective inhibitory amines contained a catechol nucleus, an alcoholic hydroxyl, and an N-alkyl substituent larger than methyl (Lands and Tainter, 1953). Greatest inhibitory action was obtained with isopropyl or cyclopentynol N-substituents (Table IIIA) . A hydroxyl on the benzene ring was more effective in the meta than in the para position (Table IIID), and an alcoholic hydroxyl was more important for inhibitory action than either one of the phenolic groups (Table IIIB). The effect of increasing the size of the side chain of compounds in which the N-substituent was isopropyl, cyclopentynol, or cyclohexyl was compared with unsubstituted compounds (Table I I I C ) . Highest inhibitory potency was obtained with the 2-carbon side chain; potency diminished in the order of C-2 > C-4 > C-3 > C-5. The 4carbon was more favorable than the 3-carbon for inhibitory action, in contrast to pressor potency which declines in the order C-2 > C-3 > C-4 (Lands e t al., 1950; Tainter et al., 1934). It would be interesting to test substances with large side chains but

TABLE I1 EFFECT OF SUBSTITUTION IN PHENYLETHYLAMINE MOLECULE^ Substitution in the phenyl nucleus

&Carbon substitution

a-Carbon substitution

N-Alkyl substitution

Hydroxyl: m-Hydroxy more active than phydrosy- Hydroxyl: Introduction of Methyl: This substituent In arylalkylamines ina hydroxyl group inprovides longevity of creased toxicity not prosubstituted compounds but pcompound less action. CNS excitation creases pressor activity nounced and pressor actoxic. An o-hydroxy-substituted substance is no and toxicity increased; but decreases CNS exmore active than phenylethylamine. In general, tivity reduced; in arylpressor activity dealkanolamines, N-methcitation and toxicity activity is greatest with the substituted phenylcreased. Introduction of ylation decreases prespropanolamines than the phenylethylamines Alkyl: Introduction of higher alkyl homologs sor activity and in.41kyl: Introduction of an alkyl group into the methyl group decreased leads to inactivity creases toxicity. Diphenyl nucleus increases the toxicity and norCNS excitation and alkylation, or convertoxicity. As size of almally lowers the pressor potency. The methyl sion into a tertiary group, in o-, m-, or p-positions decreases the ackyl substituent inarnine, decreases pressor tivity of 8-phenylethylamine, and larger alkyl creases, pressor activity activity; the larger the decreases and toxicity groups magnify this adverse effect; if the radical is large enough, aa benzyl, the compound beincreases alkyl substituent the greater the decrease. If comes depressor. The m- and p-methyl-substiKetone: Slight activity the N-alkyl group is tilted phenylpropanolamines are more toxic than or inactive large enough the molethe parent substance, the m-substituted being cule may take on anesmore toxic than the p-substituted amine thetic properties. QuaHalogen: Introduction of a fluorine atom into the ternary ammonium phenylalkylamines increased toxic properties; compounds have curareintroduction of fluorine atoms in the 0-, m-, or like actions p-positions in phenylpropanolamine decreased pressor potency in the order given Methoxy : Introduction of methosy groups, regardless of position, increased toxicity and derreased circulatory effects Data summarized to 1945 (from Hartung, 1945).

B

5i E*

TABLE I11 hIODIFICATION OF PHENYLETHYLAMINE hIOLECL7LE FOR OPTIM.\L INHIBITORY SYMP.\THOMIMETIC ACTIVITY" Molecule

A.

H o ~ c H . o H . c H . . ~ . R

Modification

Test

La m

Substituents and Potencies:

R: N-Substituent

> 0

Bronchodilation Pbency:

Cyclopentyl Isopropyl

2

1

Ethyl

Amy1

Propyl

Methyl

H

1/4

1/6

I/0

1/64

1' 4

iLa5

2 m

HO OH

OH

Modification

of carbinol

Vasdepression

group

1/200

OH

OH

H

H

H

H

H

OH

H

H

H

OH

H

H

H

OH

OH

1/800

1/1000

Inac-

Pressor

1/300 1/500

=O

tive Isopropyl Varying length of side chain

R = H: Bronchodilation

HO

CH.OH.CH..NH.CH(CH,j,

B~~~~~~~~~

Bronchodilation

Cyclopentyl

Cyclohexyl

1.5-2

1/10

< 1/1000

1/100

moo

1/ 7

1/100

H 1/15

R = CH,: R = C,H,:

< 1/1000 1/2.5

1

R = C,H,:

c 1/1000

1/1000

R:

OH

OH

H

H

K:

OH

H

1

1/00

OH 1/400

1/8W

Potency:

< 1/1000

< 1/1000

H

204

E. MARLEY

without ring hydroxyls (see Table IIIC). Belleau (1961) suggested that inhibitory activity is a property inherent in the catechol hydroxyl groups rather than in the large cationic head. The force of attraction between two oppositely charged ions is greater when the radius of the ion is smaller. The large side-chain substituent increases ion radius, so there would be a decrease in its binding constant; there would also be steric hindrance a t the cationic head. This would impair ion-pair formation a t the anionic site. If Belleau’s view is correct, increasing bulk of the side chain would diminish excitatory potency but in the absence of ring hydroxyls i t would not increase inhibitory properties. Ariens (1961) tested a homologous series of N-substituted noradrenaline derivatives. Inhibitory activity did not increase with a larger number of carbon atoms in the substituents on the nitrogen atom as in the molecules tested by Lands and Tainter (1953). Sympathomimetic action was lost but a new property emerged; there was a gradual change in the series from agonist (noradrenaline) t o competitive antagonist (phenyl isobutylnoradrenaline) . AriEns proposed that in the receptor vicinity there were indifferent structures which participated in the interaction with the mimetic; although there was loss in binding energy for the specific receptors, the additional nonspecific binding forces compensated for the loss of affinity. However, the introduction of phenyl rings a t a suitable distance from the specific parts in the original a g o n i s t a s a rule three to four interatomic distances-led to an increase in affinity to the specific receptors. An important contribution as well as an important departure in technique came from Vane (1961). The amines were tested for excitatory and inhibitory activity on one tissue, rat stomach strip (Vane, 1957). The amines fell into three distinct groups: (1) those which relaxed the tissue; (2) those which had a biphasic effect; and (3) those which produced a contraction. The three groups were chemically distinct. Only catecholamines produced relaxation and this was presumed to be through a combination with receptors for adrenaline or noradrenaline (Table I V ) . Compounds with a hydroxy group either on the phenyl nucleus or on the p-carbon atom of the side chain produced the biphasic reaction, whereas compounds with an unsubstituted phenyl nucleus or with methoxy, chloro, or methyl substituents contracted the tissue. The sympathomimetic amines which contracted the r a t stomach strip were antagonized by bromolysergic acid by phenoxybenzamine, and by tryptamine desensitization, Prolonged exposure of the rat stomach strip to amphetamine, mescaline, p-phenylethylamine, etc., desensitized the tissue to the actions of tryptamine or 5-HT. Vane (1961) concluded that the amines which contracted the stomach strip did so by combining with tryptamine re-

ADRENERGIC SYSTEM AND SYMPATHOMIMETIC AMINES

205

TABLE IV CL.A.SSIFICATION OF SYMPATHOMIMETIC AMINESACCORDING TO RESPONSE OF RAT STOMACH STRIP" Tryptamine-like activity (contraction of rat stomach strip)

Compounds with mixed effects (biphasic response of rat stomach strip)

Noradrenaline-like compounds (relaxation of stomach strip)

8-Phenylethylamine (+)- and (-)-Amphetamine Mescaline Methylamphetamine Pipadrol Phenmetrazinc

Tyramine ( - )8-Phenylethanolamine Hydroxyamphetamine Hydroxyephedrine

Dopamine (+) and (-)-Noradrenaline Cobefrin (-)-Adrenaline Epinine Isoprenaline Dihydroxyephedrine

I

a

From Vane ( 196 1).

ceptors as defined by Gaddum (1953). The findings were applicable to other types of smooth muscle. Greenberg (1960a) tested a series of amines on the heart of Venus inercenaria; they fell into two groups. Whereas the catecholamines were excitatory, the response was not obtained with phenylethylamine, tyrarnine, ephedrine, mescaline, histamine, nor with the basic N-alkylamines. Phenylethylamine and tyramine had 5-HT properties. The simplest structural requirement for 5-HT activity was a flat aromatic nucleus with a 2-aminoethyl side chain. Two receptor regions could be specified: a flat surface area 11 A by 9 A complementary to the benzene or indole ring and a contiguous ovoid depression 6 A by 4 A and up to 3.5 A deep, which accepted the terminal amino groups of the tryptamines or phenylethylamines (Greenberg, 1960b). Receptor attributes for Venus mercenuria would appear similar to those for mammalian tissues. The aromatic nucleus would be bound to the receptor by weak unspecific van der Waal's forces, with hydrogen bonding of the terminal amino group to some negative site in the receptor. BETWEEN ADRENERGIC RECEPTOR AND B. DIFFERENTIATION ADRENERGIC NEURON

The conception of the sympathomimetic amines derived from the work of Barger and Dale (1910) was of a group of similarly acting substances but with quantitative differences in activity. An indication that the view needed modification came from the observation that cocaine abolished the pressor action of tyramine (Tainter and Chang, 1927) but enhanced that of adrenaline (Frohlich and Loewi, 1910) ; this suggested that adrenaline and tyramine differed qualitatively in action.

206

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Increased sensitivity to some and decreased sensitivity to other sympathomimetic amines is found after postganglionic sympathetic denervation of smooth muscle; the denervated radial muscle of the cat's iris becomes supersensitive to adrenaline ( Meltzer, 1904) and to noradrenaline (Burn and Hutcheon, 1949), but virtually insensitive to ephedrine or tyramine (Burn and Tainter, 1931; Burn, 1932). It seems there are two groups of amines: those like ephedrine or tyramine depending upon the integrity of the postganglionic sympathetic nerves for their action and those like adrenaline with enhanced action after tissue denervation. Denervation has similar effects to cocaine, increasing sensitivity to catecholamines and decreasing the binding of amines to tissues (Hertting et al., 1 9 6 1 ~ ) The . diminished binding may be a t nonspecific receptor sites, as denervation causes an increase in the receptor area (Axelsson and Thesleff, 1959), a t least for acetylcholine. Bacq (1936a) compared the effect of various amines on the cat's denervated and innervated nictitating membranes; the division of the amines with the denervated iris was applicable to the nictitating membrane (Lockett, 1950). A larger series of amines was tested by Fleckenstein and Burn (1953) and classified in three groups (Table V). These TABLE V CLASSIFICATION OF SYMPATHOMIMETIC AMINES ACCORDINGTO RESPONSEOF CAT'S CHRONICALLY DENERVATED NICTITATING MEMBRANE" Little or no contrartion of the membrane* 8-Phenylethylamine Amphetamine N-Methylamphetamine p-Hydroxy-N-methylamphetamine Tyramine m-Tyramine

Diminished contraction of the membranec

Enhanced contraction of the membraned

p-Phenylethanolamine p-Hydroxy-p-phenylethanolamine Ephedrine p-Hydroxyephedrine

Adrenaline Noradrenaline Dihydroxyephedrine Cobefrin Dopamine Epinine Phenylephrine _ _ _ _ _ ~

From Fleckenstein and Burn (1953). Substances with not more than one -OH group on the benzene ring and without a -OH group on the 8-carbon atom. c Substances with not more than one -OH group on the benzene ring but having a -OH group on the 8-carbon atom. dsubstances with one or more --OH groups on the benzene ring and with a -OH group on the 8-carbon atom. 0

were (1) amines with one hydroxyl only and on the benzene ring, with little or no effect on the denervated nictitating membrane; (2) amines with one hydroxyl on the benzene ring and on the P-carbon atom, less

ADRENERGIC SYSTEM AND SYMPATHOMIMETIC AMINES

207

effective on the denervated than on the normal membrane; and (3) the catecholamines which had a much greater effect on the denervated than on the normal membrane. There is a close similarity in the behavior of the nictitating membrane to the sympathomimetic amines after denervation and after cocaine (Fleckenstein and Bass, 1953; Fleckenstein and Stockle, 1955). Later Burn and Rand (1958b) suggested that adrenalinelike amines act directly upon the tissue, whereas amines like amphetamine or ephedrine act indirectly through the release of noradrenaline. Such a classification is applicable when the effect of the amines on in vivo smooth muscle is being examined. Normally, the sympathomimetic amines excite salivary secretion. However, the amines can be divided into three groups for their effect on the sympathetically denervated submandibular salivary gland in the cat (Stromblad, 1960). While the secretory effect of P-phenylethylamine and tyramine was lost, that of adrenaline and noradrenaline was augmented. Dopamine and ephedrine represented an intermediate group, the secretory response to which was diminished but not abolished; this could be explained by assuming that the denervated gland had become sensitized to the direct effect of these compounds, but the indirect action had been abolished. That cross-tachyphylaxis developed to the indirectly acting amines suggested a similar mechanism of action for these compounds (Stromblad, 19GO). Belleau (1961) outlined a hypothesis providing for the activity of sympathomimetic amines. His ideas have been considered in the section on the adrenergic receptor. Briefly, he suggested that compounds with a small cationic head, e.g., phenylethylamine, had strong excitatory activity, whereas the catechol groups, as in adrenaline, conferred inhibitory activity and played a role independent of the cationic head. It would be difficult t o envisage antipodal effects of these two types of amines in vivo whcre the action of the phenylethylamine mimics through noradrenaline release that of the adrenaline-like amines. For example, both groups normally contract the nictitating membrane and dilate the iris. Accordingly, if many sympathomimetic amines act on smooth muscle indirectly through the release of noradrenaline, the accepted structureaction relationships need substantial reinterpretation (Vane, 1962). There is a solution to the impasse. As denervation depletes tissue stores of noradrenaline, a direct action of the amphetamine type of amine might emerge on denervated tissue, no longer obscured by local noradrenaline release. At first sight, the results with the nictitating membrane and salivary gland militate against such a suggestion, for amines normally acting through noradrenaline release were ineffective after tissue denervation. Yet there are hints of another action, for the effect of small

208

E. MARLEY

doses of tyramine on the denervated nictitating membrane was enhanced but that of large doses decreased (Bulbring and Burn, 1938; Lockett, 1950). Enhanced sensitivity to small doses of tyramine and p-hydroxyN-methylamphetamine may persist for weeks after membrane denervation (Fleckenstein and Burn, 1953). The persistant effect may also be due to incomplete denervation, for even after removal of the stellate and superior cervical ganglia the isolated nictitating membrane may respond to excitation of its nerves (Burn et al., 1959). It may also be due to reinnervation by collateral sprouting (Murray and Thompson, 1957). The mydriatic action of the sympathomimetic amincs is customarily studied with the iris constricted by light; consequently its response is the resultant of the dilator effect of the amines and its reaction to light. After postgnnglionic sympathetic denervation of the iris (Table VI) the CLASSIFICATION

TABLE VI SYMPATHOMIMETIC AMINESACCORDING TO RESPONSE OF CAT’S IRIS CHRONIC SYMPATHETIC POSTGANGLIONIC DENERVATION~

OF

AFTER ~

~~

Mydriatic action lost* 8-I’henylethylamine Amphetamine Phenmetrazine Methyl phenidate 2,5-Dimethoxyphcnylethylamine 3,5-I)ime thoxyphenylethylamine

Mydriatic action diminishe& Tyramine Pholedrine Ephedrine @-(3,5-Dimethoxy)phenyl8-hydroxyethylamine

Mydriatic action enhancedd Adrenaline Noradrenaline Cobefrin Isoprenaline Dopamine Epinine Phenylephrine Oxedrine Metanephrine Normetanephrine

From Marley (1962).

* Phenylethylamines without -OH

groups. Substances with a -OH group on either the benzene ring or on the &carbon atom of the side chain. d Substances with -OH groups in the 3,4-position (with or without a 8-OH on the side chain), or with one -OH group on the benzene ring together with a -OH group on the p-carbon atom. c

sympathomimetic amines can be divided into (1) phenylethylamines without -OH groups, the normal mydriatic action of which is lost; (2) substances with an -OH group either on the phenyl radical or on the P-carbon of the side chain to which the iris becomes less sensitive; and (3) substances with -OH groups in the 3,4-position on the phenyl radical, or one -OH group on the benzene ring and on the p-carbon to which the denervated iris is supersensitive (Marley, 1960a). These three groups correspond approximately with those of Fleckenstein and

ADRENERGIC SYSTEM AND SYMPATHOMIMETIC AMINES

209

Burn (1953). Amines, which in their experiments had no action on the denervated nictitating membrane, were ineffective on the denervated iris; amines to which the nictitating membrane was supersensitive the iris responded to with exaggerated sensitivity. The experiments excluded a direct mydriatic action of the phenylethylamines on the denervated iris; a direct miotic action was not precluded as the iris was initially constricted. If the sympathetic denervated iris was first dilated by dark adaptation or by acute or chronic parasympathetic denervation, phenylethylamine and tyramine were, surprisingly, found to constrict the iris (Fig. 3) ; the catecholamines remained mydriatic

FIG.3. Change in response to tyramine after chronic sympathetic denervation of the iris. A-D. Diminished mydriatic action; E H . Appearance of miotic action. A-D. Chloralosed cat, 2.0 kg ; acute bilateral adrenalectomy. Right superior cervical and vagal nodose ganglia removed 11 days previously. A and C. Controls. B. Dilatation of innervated but not of denervated iris 30 seconds after tyramine (0.5 mg/kg, iv). D. Greater mydriatic action of tyramine (3.0 mg/kg, iv) on innervated than on dcnervated iris. E-F. Chloralosed cat, 2.1 kg ; acute bilateral adrenalectomy. Right superior cervical and vagal nodose ganglia removed 18 days previously. Hexnmethonium bromide (1.0 mg/kg, iv) given in prior 15 minutes. E. Controls. F. Dilatation of innervated but constriction of denervated iris 30 seconds after tyramine (2.0 mg/kg, iv). G-H. Chloralosed cat, 1.5 kg; acute bilateral adrenalectomy. Right superior cervical and vagal nodose ganglia removed 20 days previously and bilateral removal of the ciliary ganglia 8 days previously. G. Controls. H. Contraction of the doubly denervated right iris and dilatation of the parasympatheticdenervated left iris 45 seconds after tyramine (1.5 mg/kg, iv).

210

E. MARLEY

(Marley, 1962). The phenylethylamine type of amine thus has a direct action on the tissue quite independent of any indirect action through noradrenaline release. Tyramine had a direct action on the cocainized isolated arterial strip (Furchgott, 1961b); it therefore behaved as a partial agonist a t the receptor, although its main action was indirect. The antipodal effect of the two groups of amines on the iris was reminiscent of the excitatory and inhibitory actions proposed by Belleau (1961). Other suggestions by Belleau with respect to structure and activity become applicable, such as the impairment of excitatory activity by steric hindrance a t the cationic head. Thus tyramine and phenylethylamine were miotic, whereas the structurally similar amines pholedrine and amphetamine had feeble or no miotic properties. This could be ascribed to steric hindrance a t the cationic head by their amino and/ or a-carbon substituent which would lift the cationic head away from the receptor, decreasing the strength of the interaction. The miotic action of the phenylethylamine type of amine remained after chronic parasympathetic denervation of the iris or hyoscine but was reduced, not abolished, by the tryptamine antagonist bromolysergic acid; it is possible that these amines activate tryptamine receptors in the iris. Mydriatic potency on innervated and denervated irides varied with structure. An amine with phenolic hydroxyls in the 3,4-position (Epinine) was more potent than one with hydroxyls in the 3- or 4-position with (phenylephrine) or without (tyramine) a hydroxyl on the P-carbon atom. Amines with a phenolic hydroxyl in the 3-position (phenylephrine) were more potent than those with the hydroxyl in the 4-position (oxedrine). The iris was supersensitive to these compounds; the catechol nucleus is not, therefore, essential for sensitized responses as noted by Drake et al. (1939). Blaschko (1950) pointed to the similar potency of (+)-adrenaline and of Epinine. He suggested that the similarity was explained by the position of the -OH group in (+)-adrenaline, which was unable to exert any effect on the combination between (+)-adrenaline and the receptor ; (+)-adrenaline therefore behaved like Epinine. The importance of the P-hydroxyl would explain the greater mydriatic potency of adrenaline and noradrenaline than of Epinine and dopamine, amines otherwise structurally identical. Moreover, a compound with a hydroxyl on the P-carbon and in the 3-position on the benzene ring (phenylephrine) was just as potent as an amine with 3,4-phenolic hydroxyls only (Epinine). A hydroxyl on the &carbon was more important for mydriatic action than a hydroxyl in the 4-position on the benzene ring. Thus, ephedrine was more effective than tyramine or pholedrine, and retained mydriatic

ADRENERGIC SYSTEM AND SYMPATHOMIMETIC AMINES

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action on the denervated iris when tyramine had either no, a biphasic, or a miotic action. As the threshold dose for ephedrine was raised by denervation, the substance presumably dilated the iris by both direct and indirect actions. Substitution on the NH, group also modified mydriatic activity. N-Methylated compounds were more potent than unsubstituted amines (adrenaline, Epinine, metanephrine more effective than noradrenaline, dopamine, and normetanephrine, respectively). The structure-activity classification applied only to the anesthetized animal. In the intact conscious cat or the enckphale isole' preparation, phenylethylamine or amphetamine dilated instead of being ineffective or contracting the sympathetic denervated iris ; as dilatation occurred in denervated and innervated irides it could not be due to the nicotine-like action of amphetamine on-4he superior cervical ganglion described by Reinhert (1961). When the animal was anesthetized the dilatation disappeared, suggesting that in the normal animal the mydriatic action of the amines is partly central in origin due to inhibition of parasympathetic tone (Marley, 1961b) . Thus, mydriasis produced by the p-phenylethylamine type of amine seems to be the resultant of indirect dilator action elicited both by central inhibition of parasympathetic pupil constrictor tone and by peripheral excitation of the sympathetic nerve endings in the iris, overwhelming direct activation of the sphincter pupillae. BETWEEN ADRENERGIC RECEPTOR, C. DIFFERENTIATION ADRENERGIC NEURON,AND/OR CHROMAFFIN CELL

Reserpine modified response to sympathomimetic amines ; i t enhanced the pressor action of noradrenaline (Bein et al., 1953) but abolished that of tyramine (Carlsson et al., 1957b). The sympathomimetic amines could be divided into three groups (virtually identical with those of Fleckenstein and Burn, 1953; Vane, 1961; Marley, 1962) for their effect on blood pressure in dogs pretreated with reserpine (Maxwell et al., 1959). Thus pressor action of amines with no hydroxyls or a single hydroxyl on the benzene ring was lost, that of amines with a single hydroxyl and on the p-carbon atom diminished, and that of amines with hydroxyls in the 3,4-position on the benzene ring or in the 3-position on the ring with a p-carbon hydroxyl enhanced (Table VII). As reserpine lowers blood pressure, enhanced action was defined as increase in the pressor response and not necessarily an increase in maximal pressure attained. The finding points again to the fundamental threefold grouping of sympathomimetic amines. The altered peripheral response to sympathomimetic amines may initially be a consequence of a central action of reserpine. It was first

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Pressor effect lostb

Pressor effect reducedc

Pressor effect enhanced or unaffectedd

~~

~

Phenylethylamine Amphetamine Vonedrine Methylamphe tamine Tyramine Paredrine Pholedrine

Adrenaline Noradrenaline Cobefrin Epinine Phenylephrine Oxedrine

Ephedrine Propadrine

.

.

Substances with not more than one -OH group on the benzene ring and without a -OH group on the 8-carbon atom. c Substances with a single -OH group on the @-carbonatom of the side chain. Substances with -OH groups in the 3,4-position (with or without a P-OH on the side chain), or with one -OH group on the benzene ring together with a -OH group on the &carbon atom.

thought that reserpine diminished sympathetic nervous activity; in fact, there was increased activity in preganglionic cervical sympathetic nerves (Iggo and Vogt, 1960) although the postsynaptic adrenergic neurons ceased to transmit impulses when their noradrenaline stores had been depleted. The increased pressor action of adrenaline and noradrenaline developed immediately after reserpine; as i t was not obtained after spinal cord section a t C.l, sensitization was deduced to be mcdiated centrally (Bein e t al., 1953). I n the rabbit there is a more than 50% reduction of noradrenaline in the superior cervical and solar ganglia within 1 hour FIQ.4. Development of sensitivity to catechol-like amines after reserpine administration; sensitivity persists after section of brain stem or brain destruction (A-H). Self-induced sensitization with catechol-like amines (I-K) A-E. Chloralosed cat, 3.0 kg. A : Control pressor effects of noradrenaline (NAd, 2 pg/kg, iv), adrenaline (Ad, 2.0 pg/kg, iv) and Epinine (Ep, 30 pglkg, iv). Pressor action enhanced 25 (B) and 75 minutes (C)after reserpine (1 mg/kg, iv). Increased sensitivity persists after midbrain section (D), spinal cord section at (2.1 and vagal section (El. F-H. Chloralosed cat, 2.1 kg, F. Control pressor effects of adrenaline (AD, 3 pglkg, iv) and Cobefrin (COB, 15 pg/kg, iv). Pressor action enhanced 90 minutes ( G ) after reserpine (1 mg/kg, iv). Increased sensitivity to adrenaline persists after brain destruction (H), I-K. Chloralosed cat, 3.3 kg. I. Control pressor action of Epinine (Ep, 20 pg/kg, iv), adrenaline (Ad, 3 pglkg, iv), and noradrenaline (NAd, 3 pg/kg, iv). Gradual increase in pressor action (J-K) associated with repeated injection of these amines. Time marker, minutes.

.

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of giving reserpine; maximum effect is obtained in 4 hours (Muscholl and Vogt, 1958). As the peripheral action of reserpine develops so rapidly, the sensitization to catecholamines while initially mediated centrally may soon be determined by a combination of central and peripheral or, predominantly, peripheral reserpine actions. This development can be followed (Fig. 4). Section of the midbrain and of the spinal cord a t C.l (140-160 minutes) or brain destruction (120 minutes) after reserpine, which would isolate central from spinal cord sympathetic centers, did not abolish the exaggerated pressor action of the amines; peripheral sensitizing factors were presumably operative. I n these experiments there was usually increase in both pressor response and maximal pressure attained. Other sensitizing factors may be involved, as even without reserpine the pressor action of catecholamines increased on repeated injection (Fig. 41-K) , reminiscent of the self-induced sensitization produced by adrenochrome and polyphenols (Bacq et al., 1951). Other effects of sympathomimetic amines have been tested in reserpinized animals. Phenylethylamine, ephedrine, (+) -phenylethanolamine, and tyramine, in addition to their lost pressor action, were ineffective on the cat’s nictitating membrane and spleen (Burn and Rand, 1958b). It was on the basis of such experiments that Burn and Rand (195813) suggested that amines like tyramine act in the normal animal by releasing a noradrenaline-like substance which exerts the pressor and constrictor effects; the catecholamines act directly on the tissue. (-) Phenylethanolamine retained a good deal of its action on the blood pressure and nictitating membrane after reserpine. Burn and Rand concluded that this type of compound stood midway between the “noradrenalinereleasers” like tyramine and the direct-acting adrenaline-like substances ; this type of amine corresponds with the intermediate group of amines in the other classifications. Burn and Rand (1959) pointed to the similar response of tissues denervated by degeneration of their sympathetic supply or treated with large doses of reserpine. Absent pressor response to some sympathomimetic amines and supersensitivity to others (Burn and Rand, 1958b) FIG.5. Sensitivity to catechol-like amines not fully developed in chronically reserpinized cats, although pressor action of tyramine lost; sensitivity enhanced by cocaine. A and B. Chloralosed cat, 3.5 kg; acute bilateral adrenalectomy. Pretreated with reserpine (0.5 mg/kg intraperitoneally for 2 days and 1.0 mg/kg the third day). Pressor action of Cobefrin and Epinine in chronically reserpinized cat greater after cocaine. C and D. Chloralosed cat, 3.1 kg; acute bilateral adrenalectomy. Pretreated with reserpine (2 mg/kg intraperitoneally for 2 days). Pressor action of phenylephrine in chronically reserpinized cat enhanced by cocaine, Time marker, minutes.

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and supersensitivity of the cat’s iris and spleen (Burn and Rand, 1959) occurred after chronic administration of reserpine. The absent mydriatic action after reserpine of phenylethylamine or tyramine resembled the effect of denervation; the response to the catecholamines differed from that of denervated irides. If reserpine produced all the changes of chronic sympathetic denervation in an animal so treated there should be no diffcrence between innervated and denervated irides. If the full picture of supersensitivity were taken into account (lowered threshold, shorter latency for response, greater amplitude, and duration of response), supersensitivity with reserpine appears to be incomplete (Marley, 1962). In most of the reserpine experiments quoted, fairly large doses were given. The cat pretreated with 1 mg/kg or more of reserpine has been considered an unsatisfactory experimental tool (Withrington and Zaimis, 1961). They found the heart to be in failure, sensitivity of the peripheral vessels to catecholamines to be decreased, and that the blood pressure changes were “secondary to changes in heart contraction.” Both reserpine and cocaine cause supersensitivity of sympathetic innervated organs ; reserpine releases catecholamines from tissue stores, whereas cocaine prevents their release and uptake. Although acting by different mechanisms, both drugs produce a similar end result (Whitby et al., 1960). As with denervation, the supersensitivity developed with cocaine seems more complete than that obtained with reserpine. In cats in which reserpine virtually abolished the pressor action of tyramine (Fig. 5A, C), sensitivity to the catechol and allied amines was not fully developed and could be enhanced by cocaine Fig. 5B, D) . Consequently, although the threefold division of the sympathomimetic amines can be obtained in tissues treated with reserpine, the delineation is not so sharp as in the experiments with isolated tissues, nor with in vivo experiments on denervated tissues. VIII. Sympathomimetic Amines a n d the Central Nervous System

A. SYMPATIIIN IN BRAIN Vogt (1954) in classic experiments studied the distribution of sympathin in the dog’s brain. Although sympathin was found in all parts of the central nervous system, its distribution was uneven, some parts containing a t least twenty times as much as others. The highest concentrations were in the regions which contained the diencephalic, mesencephalic, and bulbar representations of sympathetic activity; the average amount of hypothalamic noradrenaline was 1 pg/gm in the dog and 1.4 pg/gm in the cat. There was also a high concentration in the area postrema which is not nervous tissue proper; other constituents of nervous

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tissue like 5-HT and substance P (Euler and Gaddum, 1931a) are also concentrated in this ridge of tissue (Amin et at!., 1954). The substances are probably not formed there but taken up from the circulation. Gaddum and Giarman (1956) studied the distribution of the decarboxylase which forms 5-HT and probably dopamine but none was present in the area postrema. Sympathin was virtually absent from other areas of the brain and spinal cord. The sympathin concentration does not parallel the cellularity of the tissue. The very cellular cerebellar and visual cortex contained only 7 and 4%, respectively, of the sympathin in the hypothalamus (Vogt, 1957) ; since the vascularity and, therefore, presumably the vasomotor nerve supply are largely determined by cell density, the low sympathin concentration in regions of high cellularity made it improbable that all brain sympathin is disposed in vasomotor fibers as once advocated. The presence of sympathin was not linked with that of amine oxidase, nor did the distribution of amine oxidase suggest that its main role might be the destruction of sympathin. The distribution of Raab’s encephalin, described as a sympathomimetic amine differing in its chemical and biological properties from known catechol derivatives, bore no resemblance to that of sympathin (Raab and Gigee, 1951); high concentrations occurred in the cortex, the basal ganglia, white matter, and cerebrospinal fluid, tissues which contain minimal amounts of sympathin. Dopamine is also present in the brain (Carlsson et al., 1958; Montagu, 1957). No structure has been found in brain which is rich in both dopamine and noradrenaline, and the two amines seem to belong to different functional systems. It is the group of cerebral constituents with characteristic patterns of distribution and exhibiting large differences between maximal and minimal concentrations which would be expected to play a part in the specialized function of regions containing them in high concentration (Vogt, 1954). Sympathin, histamine, substance P, and 5-HT have their highest intracerebral concentrations in the hypothalamus ; this region also contains acetylcholine and vasopressin. From the point of view of the manufacture of pharmacologically active compounds, the hypothalamus is the most versatile part of the central nervous system recognized so far. The presence of sympathin in the central nervous system may be indicative of (1) a transmitter role like that assigned to sympathin in sympathetic ganglia and their postganglionic fibers, or (2) it may modify transmission similar to its modulating influence a t spinal cholinergic synapses (Bulbring and Burn, 1941; Stavraky, 1947). If it was a transmitter then it would be localized in neurons. It may, however, be in the

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glia and has indeed been found in certain gliomas (Bulbring et al., 1953). If sympathin was a central transmitter, the only neurons in which it would function would be those with nonmyelinated axons, since myelinated fibers were consistently found to lack sympathin (Vogt, 1957). It is not yet possible to state whether sympathin acts as a true transmitter, whether it modifies transmission, or whether it is rather more remotely concerned in synaptic events (Crossland, 1960). To test whether sympathin in the brain had functional significance a number of drugs which stimulate sympathetic centers were given. In addition to measuring the concentration of brain sympathin, discharge of adrenal medullary sympathin was used as a n indicator of central sympathetic activity for adrenaline release due to a direct drug action on the adrenal medulla is rare. There were species differences in response. I n cats, ether, insulin, nicotine, morphine, and P-tetrahydronaphthylamine significantly reduced adrenal and hypothalamic sympathin ; apomorphine was not consistently effective. Leptazol, caffeine, ephedrine, and ergometrine had no action on hypothalamic noradrenaline. Amphetamine diminished the noradrenaline concentration in the superior cervical ganglion and hypothalamus of the rabbit (Sanan and Vogt, 1962). In dogs, ether diminished hypothalamic noradrenaline ; morphine was ineffective. I n general, only when there was a significant fall in hypothalamic noradrenaline was there a depletion of amines from the adrenal gland. Drugs that lowered the level of hypothalamic noradrenaline also diminished that in the mesencephalon. There might occasionally be adrenal secretion without diminution of hypothalamic sympathin. While adrenal denervation would prevent medullary secretion it would not interfere with the depleting action of the drugs on hypothalamic sympathin. The relative proportions of adrenaline and noradrenaline in the depleted gland were usually the same as in the contralateral resting (denervated) gland. This suggested that stimulation of the adrenal gland by drugs acting centrally does not, as a rule, lead to preferential discharge of adrenaline (Vogt, 1954), as may occur on electrical excitation of the hypothalamus. Sympathin in the area postrema was unaffected by drugs depleting hypothalamic sympathin. The most extreme view taken of the role played by the catecholamines in the brain is that their size is directly correlated with behavior, such as excitement or motor activity. There is no evidence for this attractive hypothesis (Vogt, 1961). Administration of any drug which causes prolonged central sympathetic discharge, whether or not accompanied by signs of rage, fear or excitement, produces a fall in catecholamine concentration in the brain (Vogt, 1954). When the effect of the

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drug subsides, the stores remain low for some time, but behavior reverts to normal. Reserpine, which sedates, also reduces the brain catecholamines. Animals given monoamine oxidase inhibitors and with raised concentrations of brain noradrenaline may exhibit perfectly normal behavior (Sanan and Vogt, 1962). B. ACTIONON BEHAVIOR AND CEREBRAL ELECTRICAL ACTIVITY The effects of the sympathomimetic amines on the central nervous system depend partly on dose, molecular structure, and route of administration. Given intravenously or intraperitoneally to nonanesthetized mammals or adult birds the amines produce behavioral and electrocortical arousal. This applies to the catecholamines adrenaline and noradrenaline (Bonvallet et al., 1954; Bradley and Elkes, 1957); amines with hydroxyls in the 3,4-position on the benzene ring but lacking a /3-hydroxyl such as dopamine (Key and Marley, 1962); the phenolic amines such as tyramine (Marley and Key, 1963) ; amines with a single phenolic and alcoholic hydroxyl on the &carbon atom such as phenylephrine (Rothballer, 1957a) ; amines with a hydroxyl on the p-carbon atom such as ephedrine (H. E. Himwich, 1959) ; phenylethylamines without hydroxyls such as amphetamine (Schallek and Wala, 1953; Bradley and Elkes, 1957) and methyl phenidate and phenmetrazine (Key, 1958) ; amines with indole or 5-carbon rings such as a-methyl tryptamine and cyclopentamine (Dewhurst and Marley, 1964a) ; and aliphatic amines such as tuaminoheptane (Key and Marley, 1962). The sympathomimetic amines exert their effect on wakefulness through the brain-stem reticular formation. It is not known whether they act through localized receptors or through diffuse systems of cells. Some localization has been possible; in the rabbit, amphetamine elicits arousal by an action on the midbrain but not on the pontine or bulbar reticular areas (van Meter and Ayah, 1961). Testing drugs in this way gives little idea as to how activity varies with structure. The most that can be said is that compounds with a methyl substituent on the a-carbon are longer acting than those without, and that hydroxy substituents on the &carbon (ephedrine) or the benzene ring (tyramine) reduce central excitatory activity. From previous consideration of receptor geometry it is unlikely that such a wide structural range of amines would act on similar receptors. The series includes amines which at least peripherally act directly on the tissue or indirectly through noradrenaline release. The behavioral and electrocortical effects of the sympathomimetic amines are directly related, the alert behavior produced being associated with neocortical electrical activity found in the normal alert animal.

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In contrast, the parasympathomimetic and anticholinesterase drugs produce changes in neocortical electrical activity which do not correspond with behavior (Bradley and Elkes, 1957). The action of amphetamine may also imply another mechanism. The intense cortical arousal induced by amphetamine generates corticofugal effects which inhibit reticular activity (Dell, 1961). There is, moreover, an inverse relation between neocortical electrical activity and behavior, on the one hand, and hippocampal electrical activity, on the other (Green and Arduini, 1954). The relation holds for the effect of the sympathomimetic amines; after amphetamine, low voltage desynchronieed neocortical electrical activity was accompanied by rhythmic 2-4 cycles/second hippocampal potentials with superimposed fast activity (Bradley and Nicholson, 1962). Bradley and Nicholson (1962) suggested that the inverse relation between hippocampal and neocortical electrical activity might be due to inhibitory control of hippocampal neurons by the midbrain limbic area; electrocortical activity found during wakefulness and after amphetamine could result from activation of this inhibitory reticular mechanism.

C. DIBECT OR INDIRECT ACTIONOF CATECHOLAMINES An important issue is whether the arousal which follows intravenous injection of the natural catecholamines is a direct action on the ascending reticular formation or indirect through circulatory changes or the production of active metabolites (Vogt, 1961). Attempts have consistently been made to dissociate the central from the pressor effects of the amines. Bonvallet et al. (1954) found that the hypertension was not responsible for the arousal ; they showed discrepancies in the time course of hypertension and arousal, and arousal when blood pressure changes were negligible. This was confirmed by Rothballer (1956) ; electrocortical activation occurred in cats with midbrain coagulation in which adrenaline had either no effect on the blood pressure or produced hypotension. Rothballer also showed that the effects of noradrenaline were the same, weight for weight, as those for adrenaline in spite of different blood pressure effects. Baust et al. (1963) reinvestigated the problem. Their resulk supported the idea of an action due to the rise in blood pressure mediated through the cells of the reticular-activating system. In all probability the blood pressure was the predominant or even the only factor leading to “adrenaline-induced” arousal. Thus, electrocortical arousal after intravenous adrenaline either coincided with or occurred 1-3 seconds after the blood pressure began to rise; the latency depended on the sensitivity of the preparation to other arousal stimuli. If the blood pressurk was kept constant during intravenous injection of adrenaline, electro-

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cortical arousal was delayed and briefer than if the pressure had not been kept constant; if the blood pressure was raised by rapidly injecting saline into the aorta, electrocortical arousal was elicited but disappeared before the pressure returned to normal. In the intact animal, the picture is further complicated by the effects of adrenaline on the carotid sinus; stimulation of baroreceptors by hypertension causes inhibition of the ascending reticular-activating system, thus opposing the arousing action of adrenaline a t this site. Whether arousal or cortical inhibition is produced will depend on the interaction of opposing influences. I n the adult cat, adrenaline produced a series of alternating phases of electrocortical activation and deactivation (Rothballer, 1956). Activation was the most conspicuous and dominated the middle part of the response; the first phase of electrocortical deactivation coincided with the rise in blood pressure, while the second phase accompanied the decline of or return of blood pressure to normal. The alerting effects of adrenaline may be due to local changes in the cerebral circulation which are not reflected in the systemic circuit. Electrical stimulation of the brain which produced alerting also increased cerebral blood flow (Ingvar, 1958); a similar increase occurred with adrenaline injected intravenously. While such a mechanism would not explain all the effects of adrenaline, it might be that changes in vascular tone occur a t localized sites within the brain and that these are important. Capon (1958) has shown that vasopressin causes arousal in the rabbit and that adrenaline antagonists inhibit the central activation pari passu with the vascular effects of adrenaline. These findings led Mantegazzini et a2. (1959) to follow up some experiments by Longo and Silvestrini (1957), who failed to arouse cats by injecting adrenaline into the carotid artery. The brain-stem reticular formation is supplied by the vertebral artery ; consequently, adrenaline may not be distributed to this region if injected by the carotid route. If adrenaline produced arousal given intravenously it should do so by intraarterial injection but with a smaller dose. Mantegazzini et al. (1959) injected adrenaline or noradrenaline into the carotid or vertebral arteries. Electrocortical arousal was obtained only if the dose given was so large that the amines escaped into the general circulation ; however, intraarterial injection of adrenaline or noradrenaline may produce central vasoconstriction intense enough to counteract the usual effect of the amines. Mantegazzini et d.suggested that the delayed arousal with the large dose of adrenaline or noradrenaline could be due to their conversion during the circulation time to metabolites which acted as central excitants. However, arousal with adrenaline was obtained just as easily in eviscerated cat enc6pMe is016

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preparations in which the liver was no longer in the circulation as in intact cats (Bradley, 1961), and large doses of the metabolites metanephrine, normetanephrine, or 3-methoxy-4-hydroxymandelic acid (VMA) given intravenously to normal cats did not affect electrocortical activity (Marley and Key, 1963). That substances which modify the effect of adrenaline on peripheral receptors alter in similar fashion its central effects has been used as an argument supporting a central action of the amines; the argument could apply just as strongly for a peripheral as for a central action. The central effects of adrenaline are potentiated by cocaine which diminishes tissue binding of the amines. Pyrogallol, which potentiates the effect of sympathetic nerve stimulation or of circulating adrenaline (Bacq, 1936b,c) by inhibition of O-methyltransferase (Axelrod, 1959), prolongs arousal produced by adrenaline (Dell, 1961) ; pyrogallol itself may cause a fall in blood pressure and electrocortical arousal (Dell, 1961). Phentolamine, which blocks the peripheral action of adrenaline on a-receptors, suppresses the circulatory and electrocortical alerting effects (Capon, 1960) ; this does not apply to all species. Dell (1961) argued for a direct action of adrenaline on reticular neurons. He showed that adrenaline produced electrocortical arousal in cats with brain-stem transection in the anterior third of the pons. In these preparations the blood pressure was normal and brain-stem regulation intact, since the vasomotor centers and their afferent and efferent projecting systems lay below the section. The ascending reticular system was disconnected from trigeminal input as well as from nervous connections with the carotid and aortic baroreceptors ; the only nervous pathways connected with the reticular core were the first, second, and third cranial nerves. Dell pointed out that although alerting with adrenaline is easily obtained in encdphale is016 preparations (cat, monkey, rabbit) such experiments were not conclusive as cortical activation may be due to improved cerebral blood flow or to altered sensory input through the intact cranial nerves. Another set of observations supports the idea of a direct action of adrenaline. Rothballer (1957b) injected 1 pg adrenaline into the brain stem. Electrocortical activation was obtained from regions of the midbrain reticular formation, which corresponded almost exactly with maps made from electrical stimulation, whereas injections into other areas were without effect. That there are neurons in this region which can be excited by mechanical or other forms of stimulation makes it difficult to say how far the effects were due to adrenaline or to the mechanical effects of the injection, particularly as saline controls were often effective. Cordeau et al. (1963) gave “microinjections” of adrenaline into the brain-stem reticular formation, producing behavioral and

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electrocortical arousal in sleeping cats; the effect was ascribed to an action on reticular neurons and not “fibers de passage.” However, although control injections of tyrode solution were ineffective, 20 pg adrenaline was injected, which would be considered a large dose even if given intravenously. The large doses may have modified results, for, whereas small amounts of adrenaline applied iontophoretically depressed cortical neurons, large doses excited neurons (Krnjevi6 and Phillis, 1963a). There are strong reasons for supposing that the alerting effect of adrenaline is not due to a direct but to an indirect action on brain-stem neurons. The catecholamines are lipid-insoluble substances which do not easily penetrate the blood-brain barrier. From experiments with tritiumlabeled adrenaline and noradrenaline it appears that the hypothalamus is only part of the brain where a significant uptake of amine occurs (Weil-Malherbe et al., 1959; Weil-Malherbe, 1961) ; even there the rate of amine uptake is slow compared with most other tissues. For technical reasons it was not possible to study uptake of amines during the critical few minutes after injection, the time when the electrocortical and blood pressure changes occur. To evaluate the central effects of the sympathomimetic amines they should be given in such a way that the blood-brain barrier can be surmounted. There are a number of methods for achieving this.

D. INTRAVENTRICULAR AND INTRACISTERNAL INJECTION OF DRUGS A method for injecting drugs into the lateral ventricle of the brain in conscious cats was developed by Feldberg and Sherwood (1953). The drugs act on a limited region of the brain; they pass quickly into the third and fourth ventricles and flood the periventricular gray matter. The effects observed on intraventricular injection can be attributed to central actions of the drugs. Absorption from the ventricles into the blood stream may occur; absorption can be excluded as the cause of the effects when they differ from those observed on intravenous or subcutaneous injection (Feldberg, 1963). The depth of penetration into the brain of substances injected into the ventricles differs greatly between various regions of the brain; substances may pass deeply into nuclear regions and even through the cerebral wall. While drug entry to the brain from the blood is largely governed by lipid solubility, entry from the cerebrospinal fluid to brain is not (Mayer e t al., 1959, 1960). The essential factor which governs the uptake of substances appears to be the neuroglia. The differences between uptake by gray and white matter and between different regions of gray matter would be due mainly to the fact that the distribution of the various glial cells varies between gray and white matter and, within

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the gray matter, between different regions (Feldberg, 1963). That the uptake of substances from the cerebral ventricles is governed by neuroglial properties accords with recent electron microscopic findings (Gerschenfeld et al., 1959; Luse, 1959; Luse and Harris, 1960), which indicate that neuroglial cells participate actively in the movement of fluids within brain tissue. When adrenaline is injected into the cerebral ventricle of the cat it penetrates probably only 2 or 3 mm deep into the walls of the third ventricle and aqueduct. The sedative action is therefore probably an action on mid-line structures in the diencephalon and perhaps also in the mesencephalon (Feldberg, 1961). The effech of intraventricular injection of adrenaline or noradrenaline are the opposite of what ie seen on intravenous injection. During the first minutes after intraventricular injection of 20-80 pg adrenaline the cat makes licking and swallowing movements followed by retching and vomiting. Within 10-20 minutes the animal becomes drowsy. The eyes are closed, but when handled or disturbed, the cat stares with open, apparently unseeing eyes (Feldberg and Sherwood, 1954) ; full recovery occurs within 3 hours. Intraventricular injection of calcium produces a similar picture (Feldberg and Sherwood, 1957). The instillation of 5-250 pg adrenaline into the lateral ventricle of psychotic &man subjects elicited drowsiness (Sherwood, 1955). The soporific action of adrenaline is by no means an isolated observation. Adrenaline produced analgesia and stupor if injected subdurally or intracerebrally (Bass, 1914). The soporific action of noradrenaline or adrenaline given into the cisterna magna or into the lateral ventricle has been found in mice, dogs, and sheep (Haley and McCormick, 1957; Leimdorfer, 1950; Palmer, 1959). The catecholamines can produce stupor given intravenously, but large doses are required. Adrenaline can pass from the blood stream into the cerebral ventricles and the subarachnoid space surrounding the brain and spinal cord (DraBkoci et al., 1960), but the amounts of adrenaline infused into the cat were greater than those that would be released under physiological conditions, DraBkoci e t d. suggested that adrenaline secretion into the blood stream may first produce alerting, but later if it passes into the cerebrospinal fluid i t acts directly on the brain producing languor. The arousal produced by adrenaline or noradrenaline injected intravenously may not, therefore, be a direct central action; the amines may initiate afferent impulses elsewhere in the body which affect mid-line structures in the brain-stem but in the opposite way to which they would be affected by adrenaline injected intraventricularly (Feldberg, 1961). It is interesting that adrenaline given intraventricularly has an opposite effect on behavior and on tremor to that given intravenously.

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Thus adrenaline or noradrenaline injected intraventricularly abolished the tremor produced by chlorpromazine or pentobarbitone sodium (Domer and Feldberg, 1960) ; ephedrine and amphetamine were ineffective. I n anesthetized cats tubocurarine perfused from the lateral ventricle to the aqueduct elicits tremor and muscle jerks. Tremor ceases when the amines are added to the perfusion fluid although the jerks persist (Feldberg and Malcolm, 1959) ; adrenaline was more effective than noradrenaline and much more potent than dopamine (Carmichael et al., 1962). The antitremor action of these amines was presumed to be mediated through structures in the hypothalamus and perhaps also the rostra1 part of the gray stratum. I n cats an intravenous infusion of adrenaline first accentuated and then abolished pentobarbitone tremor (Hall and Goldstone, 1940) ; in man intravenous infusion of adrenaline but not noradrenaline elicited tremor in some normal subjects and accentuated tremor in patients with Parkinson’s disease (Barcroft et al., 1952). The antitremor as well as the sedative effect can be regarded as depression of activity in the core of the brain stem (Feldberg, 1961). As these are regions rich in sympathin (Vogt, 1954) there may be some connection between their localization and their sites of action. It may be that physiological release of brain-stem noradrenaline is involved in the control of tremor or shivering (Feldberg, 1963). Intraventricular injection is an audacious and extremely fruitful approach. Criticism has been made that it is an “unphysiological” technique, yet it is no more unphysiological than studying isolated organs deprived of their blood supply and bathed in physiological media. If, as appears likely, drugs gain access to the brain not only through the blood- brain barrier but by secretion into the cerebrospinal fluid within the ventricles (Roth et al., 1959), then it is a logical method for study ing drug action on the central nervous system. Unfortunately, discrepancies occur between the behavioral and electrocortical patterns produced by adrenaline. During the period immediately after the intraventricular injection, drowsy electrocortical activity is found in the cat; during the height of stupor, electrocortical activation is conspicuous (Rothballer, 1959). Moreover, amphetamine and N-methylamphetamine, which readily cross the blood-brain barrier on intravenous injection and elicit behavioral and electrocortical alerting, might reasonably be expected to have this action when given intraventricularIy. Unexpectedly, they made the cat lethargic and intensified the depressant action of 5-HT (Gaddum and Vogt, 1956). The intracisternal route of injection has been used by Leimdorfer (1950). Amphetamine, phenylpropanolamine, ephedrine, and tuaminoheptane given intracisternally to dogs produced excitement, whereas

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adrenaline, N-isopropyl noradrenaline, a-ethyl noradrenaline, and oxedrine elicited sopor. This dichotomy in effect between the two groups of amines accords with their general division in activity (see Table IX).

E. AMINOACIDPRECURSORS OF CATECHOLAMINES Another method of circumventing the blood-brain barrier is to give the amino acid precursors of dopamine or noradrenaline. The acids are decarboxylated near their site of action and an effective concentration of amine is built up. However, in a large series of amino acids tested by Purpura et al. (1959) all but one were ineffective by systemic administration, indicating their inability to penetrate the blood-brain barrier, whereas they were active on topical or intraventricular application. In regions of local lesions of the blood-brain barrier, the effects produced by systemically injected drugs were the same as those on topical application. The amino acids acted primarily on axodendritic synapses (Purpura e t al., 1959). Dopa presumably crosses the blood-brain barrier as its administration leads to accumulation of catecholamines in the brain (Raab and Gigee, 1951). The massive increase in tissue levels of some amines which follows the injection of the amino acid precursors may not be accompanied by a pharmacological effect (Bogdanski et al., 1958; Kako e t al., 1960)) possibly because the amines are held in an inactive form in cells. In mice and monkeys tranquilized by reserpine, large doses of dopa have a remarkable awakening effect (Carlsson e t al., 1957a) ; Everett and Toman, 1959) ; m-tyrosine had excitatory actions in mice, rats, rabbits, and dogs, which were thought to be due either to the formation of m-tyramine by decarboxylation of the amino acid (Mitoma et al., 1957) or to increased concentration of one of the catecholamines. Blaschko and Chrurjciel (1960) tested amino acids related to 3,4-dopa for their awakening effect in mice pretreated with reserpine; L-dopa and m-tyrosine had strong excitatory actions, whereas D-dopa, 2,3dopa, 2,5-dopa, and 3,4-dihydroxyphenylserine were ineffective. As there was always a delay between amino acid administration and onset of action, and D-dopa, which is not a substrate of the decarboxylase, was inactive, the amino acids were presumed to be converted to the corresponding amines. The paradox then arises that amino acid precursors of catecholamines are central excitants, whereas their products the catecholamines, given by methods that circumvent the blood-brain barrier, are central depressants. The dilemma can be resolved if amino acids have a direct action on neurons; amino acids have excitatory or inhibitory direct actions on cortical (Purpura e t at., 1959; Krnjevid and Phillis, 1961) and spinal

ADRJCNERGIC SYSTEM AND SYMPATHOMIMETIC AMINES

227

neurons (Curtis and Watkins, 1960). Activity depended on interaction of the amino acid molecules with membrane structures of definite shape, size, and charge distribution ; the membrane permeability change by which the action of an amino acid was mediated was the direct consequence of the transient interaction (Curtis and Watkins, 1960). The “amino acid receptor” could be considered to consist of an arrangement of fully ionic or partially charged atoms or groups located within a specific region of the molecular framework of the membrane. The structural requirements were elucidated. Excitatory activity was associated with one basic and two acidic groups on the amino acid; depressant action with one acidic and one basic group. The optimum distance between the amino group and acidic groups was two or three carbon atoms in depressant and excitant molecules (Curtis and Watkins, 1960). With this clear demonstration of receptor sites, the central effects produced by precursors of catecholamines given intravenously may be considered to be due to the pharmacological properties of the particular amino acids ; their conversion to the corresponding amines will complicate the picture. In support of this, L- and DL-dopa applied by micropipettes excited cortical neurons, whereas dopamine, noradrenaline, and adrenaline depressed cortical neuronal discharge initiated by synaptic activity or by the application of L-glutamate (KrnjeviE and Phillis, 1963b).

F. ACTIONOF SYMPATHOMIMETIC AMINESIN IMMATURE ANIMALS I n the immature brain the blood-brain barrier is absent or not fully effective (Bakay, 1956; Lajtha, 1957). By comparing the central effects of the sympathomimetic amines in the immature with the mature animal the influence of the blood-brain barrier could be determined. Techniques were developed for recording behavior and electrocortical activity (Key and Marley, 1961a, 1962) and for anesthetizing newborn animals for the implantation of cannulae, cortical, and muscle recording electrodes (Marley and Payne, 1962). The young and adult of three species were tested and the chicken proved the most informative (Key and Marley, 1961b). The behavioral effecb of a number of amines have been tested in birds (Clymer and Seifter, 1947; Zaimis, 1961). I n the 1- to 28-day-old bird the sympathomimetic amines had different effects (Table VIII). Amines with hydroxyls in the 3,4-position on the benzene ring with or without a hydroxyl on the &carbon atom or with a hydroxyl in the 3-position on the ring and on the /3-carbon atom (group 1) produced behavioral and electrocortical sleep (Key and Marley, 1961b, 1962) ; the 0-methylated substances metanephrine and normetanephrine inconsistently produced sleep. The bird stood or squat-

228

E. MARLEY

TABLE VIII FUNCTIONAL AND STRUCTURAL GROUP IN^ OF SOME SYMPATHOMIMETIC AMINEST E a T n D ON BEHAVIOR AND ELECTROCORITCAL ACTIVITY IN T H E 1- TO 2 8 - D A T - O L D CHICKENO ' Compound

Group

Structure

H f i 7 C - c r - T

OH

OH OH

H

OH

OH

H

OH

H

OH OH

CH,

H

H

OH OH

OH

H

H

H

CH,

Dopamine

H

OH

OH

H

H

H

H

(-) - Phenylephrlne

H

H

OH

H

OH

CH,

(i)

- Oxedrine

H

OH

H

OH

(i)-Metanephrine

H

H OH CH,O

H H

H

OH

H

CH,

(i)-Normetanephrine

H

OH CH,O

H

OH

H

H

(i)-Pipadrol

H

OHand

cP CH*-

CH,

H H

(-)-Adrenaline (-) -Noradrenaline (i)-1soprenaline

(*)-Coberrin

1

Epinlne

H H

OH

H

H

H

H

OH OH

C8H11

H

H H

CH, H CH(CH,),

CH,

CIHI

(-)-Ephedrine

H

H

H

H

OH

CH,

(i)-Phenylpropanolamine

H

H

H

H

OH

CH,

OH

CH,

H

CH, H

CH,

(i)-Methoxamine

2

(i) -Hydroxymethylamphetamine

Tyramlne (i)-Hydroxyamphelamine

0-Phenylethylamine (+)-Amphetamine

- Phenmetrazine

(i)

(i)-Methyl phenidate

3

CH,O

H CH,O

H

CH, H

H

O

H

H

H

H

O

H

H

H

H H

H

O

H

H

H

H

CH,

H

H

H

H

H

H

CH,

H

CH2 I CH,-

CIHl CH,

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

CH, - - c H ~ , - N H ,

(i)-a-Methyltryptamine

-

(i) Cyclopentamine

(i)-Tuamlnoheptane a

From Key and Marley (1902); Dewhurst and Marley (1964a).

ted as in normal sleep and behavioral and electrocortical arousal could be obtained with sensory stimuli. The central depressant action was therefore readily reversed and postural reflexes retained. Whereas similar electrocortical changes were produced by the central depressants halothane or pentobarbitone, electrocortical arousal could not be elicited and the postural reflexes were lost.

ADRENERGIC SYSTEM AND SYMPATHOMIMETIC AMINES

229

Phenylethylamines without hydroxyl groups (group 3) produced behavioral and electrocortical arousal. The benzene ring was not essential for alerting as central excitation was produced by the aliphatic amine, tuaminoheptane, and by amines with indole or 5-carbon rings (Table VIII) . Whereas electrocortical alerting was consistently produced the effect on behavior varied, increased activity and cheeping being supplanted, particularly with large doses, by ataxia. Ataxic phenomena are found in most species with large doses of the amphetaminelike amines but curiously are infrequent in man (Marley, 1960b). The suggestion that amphetamine acts on central tryptamine receptors (Vane, 1961) was supported by the similar response to amphetamine and amethyltryptamine (Dewhurst and Marley, 1964a). Amines structurally intermediate between the two groups with one hydroxyl in the 4-position on the benzene ring or on the 8-carbon atom (group 2) had no or equivocal effects; this could not be attributed to their brevity of action for, of the five tested, four had a methyl group on the a-carbon (Table VIII). The central could be dissociated from the pressor effects of the amines for, whereas adrenaline or phenylephrine produced behavioral and electrocortical sleep, equipressor doses of tyramine were ineffective. These three groups conform generally with the suggestion of Belleau (1961) that sympathomimetic amines with an unsubstituted benzene ring and small cationic head are excitatory, whereas the catechol ring confers inhibitory properties. The division of the amines into three groups is also similar to structure-activity findings with in vivo denervated mammalian smooth muscle (Fleckenstein and Burn, 1953; Marley, 1962), or in vitro mammalian smooth muscle (Vane, 1961). To this extent the geometry of central receptors in the young chicken resembles receptors in mammalian smooth muscle; it is usually possible to predict from the structure of the amine its effect in the young chicken. The activity on central receptors of an amine relative to others within the same group parallels, with certain exceptions its relative potency in peripheral receptors. Epinine is one of the exceptions, for i t is more potent than dopamine on the cat’s denervated iris but less potent in the chicken’s central nervous system. This may be because dopamine is a normal cerebral constituent (Carlsson e t al., 1958) and presumably plays some role in cerebral function, whereas Epinine is not known to be. The most potent depressant molecules had hydroxyls in the 3,4-position on the benzene ring and a hydroxyl on the 8-carbon atom. As with mammalian smooth muscle, an amine with a phenolic hydroxyl in the 3-position (phenylephrine) was more potent than one with the hydroxyl in the 4-position (oxedrine). The importance of the 8-hydroxyl was evident from the greater potency of adrenaline and noradrenaline than

230

E. MARLEY

Epinine and dopamine, amines otherwise structurally identical. P-Hydroxylation rendered an excitant molecule less active or inactive; thus amphetamine was excitant but phenylpropanolamine inactive. Modification of the cationic head or substitution with a group larger than methyl in excitant molecules reduced potency (phenmetrazine) ; Clymer and Seifter (1947) found N-acetylamphetamine to be ineffective in the chicken. Owing to the similar response in mammals to amphetamine-like and adrenaline-like amines, it was thought that these substances act on common central receptors. Because of their antipodal effects in the young chicken, the two groups of amines were unlikely to act on identical central receptors or systems; for the same reason, the central action of the amphetamine-like amines was unlikely to be due to a local release of norackenaline. The effect of antagonists supports the idea of different receptors. A specific tryptamine antagonist, methysergide (l-methyl(+)-lysergic acid butanolamide, UML 491), in a dose of 0.001 pmole, which did not affect the chick’s normal behavior or electrocortical activity, prevented or abolished for more than 60 minutes the effect of 1 pmole of amphetamine on cheeping, electrocortical, and electromyographic activity. Phenoxybenzamine and Hydergine, which block a-receptors, and pronethalol and dichloroisoprenaline, which block &receptors, were ineffective in equimolar dose or less in blocking the excitant effects of amphetamine or the depressant effects of the catecholamines on normal cheeping, electrocortical, or electromyographic activity (Dewhurst and Marley, 1964a). There are other reasons for supposing that the amphetamine and adrenaline types of amine act on different receptors (Table IX). These include the different behavioral responses of the chicken to substances from the two groups of amines (Clymer and Seifter, 1947; Zaimis, 1961) ; the dissimilar electrocortical effects in the chicken after brain-stem transection (Marley, 1963) ; the different behavioral response in dogs (Leimdorfer, 1950) on intracisternal injection of the amines; and differing behavioral and electrocortical effects in young kittens (Marley and Key, 1963). In experiments with cats on arousal responses produced by electrical stimulation of the brain-stem reticular formation, increasing amounts of amphetamine, injected intravenously, progressively lowered the threshold for arousal by electrical stimulation (Bradley and Key, 1958). There appeared to be an interaction between excitation produced by the electrical stimulus and that by the amine. If adrenaline acted similarly, then the same type of relation might be expected between dose and electrical stimulation; no such interaction was obtained (Bradley, 1961).

ADRENERGIC SYSTEM AND SYMPATHOMIMETIC AMINES

231

As the chicken matured its response to the catecholamines changed. Adult blood-brain barrier characteristics for the chicken were not present until about the fourth week of life (Waelsch, 1955), and from the first to the second month a transitional type of response was elicited with the catecholamines. Thereafter, all sympathomimetic amines tested, irrespective of group, elicited behavioral and electrocortical arousal as in the adult of other species (Key and Marley, 1962) ; arousal was sometimes not marked but drowsiness could only be elicited as in adult mammals after the administration of huge amounts of the catecholamines. It might be argued, because of the primitive nature of the avian brain, that electrical activity recorded from the chicken cerebral hemisphere does not correspond with that from mammals. The avian cerebral hemisphere is covered by a thin layer of cortex except for the anteromedial noncortical nuclei diffusus dorsalis and diffusus dorsolateralis which are the avian homologs of the mammalian neocortex (Kuhlenbeck, 1938). With recordings from many areas of the avian cerebral hemisphere, the electrocortical changes occurring either spontaneously or after the sympathomimetic amines were invariably directly related to those in behavior. The correspondence of the electrocortical changes in the adult chicken with those in adult mammals and the fact that the recordings were taken from the cortex justifies comparison with mammalian findings. Ascribing the change in response to the catecholamines as the chicken matured to an alteration in blood-brain barrier permeability is the most tempting of a number of possible explanations. Inasmuch as catecholamines given intravenously act as central depressants in the young chicken-as in adult mammals when the blood-brain barrier is circumvented-but as excitants after the development of adult blood-brain barrier characteristics, the argument is valid. Davson (quoted by Zaimis, 1961) considered that there is a definite blood-brain barrier but possibly less pronounced in the very young chicken. Whether the blood-brain barrier explanation is correct or not the specification of central receptor geometry in the young chicken is the closest to that for peripheral receptors yet attained. The greater permeability of the blood-brain barrier in the young animal does not affect the entry of all substances into the brain (Grazer and Clemente, 1957; Grontoft, 1954). A higher exchange rate of chloride (Lajtha, 1957; Waelsch, 1955) and greater permeability of young brains than adult brains to glutamic acid (W. A. Himwich et d., 1957), lysine (Lajtha, 1958), leucine (Lajtha and Toth, 1961), Pa*(Fries and Chaikoff, 1941), a-aminoisobutyric acid (Kuttner et al., 1961) , and thiocyanate (Lajtha, 1957) were found. A greater lability of the young barrier to y-aminobutyric acid occurs (Purpura and Carmichael, 1960).

hl

OBSERVATIONS SUQQESTING A DIFFERENTMODEOR Species

Administration

TABLE IX SITE OF ACTIONOF AMPHETAMINE-LIKE AND ADRENALINE-LIKE AMINES IN CENTRAL NERVOUS SYSTEM

Amphetamine-like aminea

Adrenaline-like amines

8 THE

References Clymer and Seifter (1947)

Large doses subcutaneously

Drowsiness or sleep with adExcitement and cheeping with amphetamine and (+)-derenaline, Cobefrin, phenyl oxyephedrine; excitement with ephrine, and ephedrine hydroxyamphetamine

Intracistemal injection

Excitement with amphetamine, phenylpropanolamine, ephedrine, and tuaminoheptane

Sopor with adrenaline, N-isoLeimdorfer (1950) propyl noradrenaline, a-ethyl noradrenaline, and oxedrine; excitement with phenylephrine

Chicken

Intravenous injection

Amphetamine increases rate of cheeping

Adrenaline and phenylephrine produce sleep

Cat

Intravenous injection

Interaction between excitation No interaction between excitaproduced by electrical etimula- tion produced by electrical tion of the brain-stem reticular stitnulation of the brain+tem reticular formation and by formation and by amphetamine adrenaline

Chicken, 1- to 2 M a y old

Intravenous injection

Excitement, cheeping, and elec- Electrocortical and behavioral Key and Marley trocortical arousal produced sleep produced by adrenaline, (1961b, 1962) by amphetamine, methyl noradrenaline, isoprenaline, Dewhurst and Marley phenidate, phenmetrazine, Cobefrin, dopamine, and (1964a) phenylephrine. Action not anpipadrol, cyclopentamine, LT tagonized by methysergide nor methyltryptamine, tuaminoby a- or &receptor blockers in heptane. Action of amphe& equimolar doses or less. The amine, a-methyltryptamine, adrenaline-like amines act as blocked by methysergide but

Chicken

Zaimis (1960s) Bradley (1961)

M

not by (I- or 6-receptor blockem in equimolar doses or leas. The amphetamine-like amines act as physiological antagonists to the adrenaline-like amines

physiological antagonists to the amphetamine-like amines

Chicken, young and adult

Intravenous injection

Electrocortical arousal elicited in bir& with brain-stem tranaection

Antagonism of electrocortical Marley (1963) arousal produced by amphetamine in 1- to 2S-day-old birds with brain-etem transection. Electrocortical arousal difficult t o obtain in adult birds with brain-stem transection

Kitten

Intravenous injection

Behavioral and electrocortical Soporific action until the thirdarousal from birth, but adult fifth week pattern electrocorticsl arousal only by third-fXth week of life

Marley-and-Key (1963)

9,

3 m

rc

Ex-

234

E. MARLEY

The reduced entry of many biologically important substances into the brain as the animal matures may be a reflection of cerebral metabolic requirements and not primarily a measure of blood-brain barrier function (Lajtha, 1957). There may be better transport mechanisms capable of restoring physiological levels faster or to a greater degree in the adult than in the newborn brain (Lajtha, 1962); brain enzyme activity and maximal concentration change before and immediately after birth (Sperry, 1962). The difference between the response in the young and adult chicken to the catecholamines could be due to faster maturation of inhibitory than of excitatory systems. However, excitatory systems are well established in the young chicken, for electrocortical and behavioral arousal with sensory stimuli and amphetamine could be obtained from the first day. On the other hand, if the adrenaline-like amines have no direct action on neurons but rather modulate activity, they might preferentially modulate inhibitory rather than excitatory activity in the young bird if inhibitory systems are the best developed. Paton (1958) considered that excitation (as opposed to facilitation) of nerve cells by adrenaline had still to be demonstrated. As amphetamine produced behavioral and electrocortical arousal in the cat and monkey encdphale isold but not the cerveau isole’ preparations it was proposed that amphetamine exerted its effect through receptors in the brain stem (Bradley and Elkes, 1957). The central site of action of the sympathomimetic amines in the chicken was investigated by testing their effect on cerebral electrical activity and behavior after acute brain-stem transection (Marley, 1963). After the transection the chicken lay immobile and asleep; electrocortical arousal on sensory stimulation was poorly sustained and occurred only with visual stimuli. In 1- to 28-day-old chickens the amphetamine-like amines produced behavioral and electrocortical alerting. Whereas there was an increase in amplitude of the slow electrocortical potentials after the catecholamines and a reduction of the electrocortical response to eye-opening, the central depressant action of the catecholamines was better seen in the restoration of behavioral and electrocortical sleep in birds first roused by amphetamine. The effect of both groups of amines was abolished by transections above the midbrain. In adult birds, after brain-stem transection, the amphetamine-like amines readily elicited electrocortical arousal. The catecholamines no longer had central depressant actions as in the young bird. The arousal threshold was raised compared with the intact adult chicken, suggesting that spinal cord sensory input was important for the arousal elicited by the catechol substances. If the transection was a t or above the level of the trigeminal nerves, arousal could not be obtained.

ADRENERGIC SYSTEM AND SYMPATHOMIMETIC AMINES

235

It appeared then that in 1- to 28-day-old birds the sympathomimetic amines produced their antipodal eff ecta on behavior and electrocortical activity through receptors or systems in the brain stem; in the adult bird, the amphetamine-like amines acted on brain-stem receptors or systems, but the alerting produced by the catecholamines was due either to their action on peripheral receptors or mediated through the lower brain stem. The results in the adult bird favor a different mode of or site of action for the amphetamine-like and the adrenaline-like amines. The young of other species tested included the guinea pig and kitten (Marley and Key, 1963). The young guinea pig is more mature than most species at birth (Windle, 1940) ; electrocortical activity (Jasper et al., 1937) and the blood-brain barrier (Waelsch, 1955) are mature. I n support of the suggestion that the effect of the catecholamines is modified by blood-brain barrier maturity, all the sympathomimetic amines including the catecholamines produced behavioral and electrocortical arousal as in the adult guinea pig and other adult animals. The kitten responded differentially depending on age. The adrenalinelike amines were without effect until the ninth-fifteenth day, when electrocortical arousal with sensory stimuli could be first obtained consistently. Paradoxically, they then evoked drowsiness, although the electrocortical pattern differed from that of the drowsy adult. Amphetamine-like amines increased activity, probably due to central and peripheral action, and modified electrocortical activity during the first 10 days of life, although the adult type of electrocortical desynchronization was not elicited by them until the third-fifth week. The lack of action until the third-fourth week of life of the structurally intermediate amines could not be accounted for by their brevity of action as the three tested had a methyl group on the a-carbon. Newborn animals differ in other ways in their response to the sympathomimetic amines. Noradrenaline and isoprenaline have calorigenic properties in newborn kittens (Moore and Underwood, 1959, 1960a,b) ; adrenaline is inactive in this respect. It was suggested that cell penetration by the amine might be the determining factor for calorigenic action. The newborn kitten differed from the adult in this respect as the calorigenic action of noradrenaline declines with age (Baum et d.,1960). It may be that the different central effects of the catecholamines in the young and adult chicken is another expression of the finding with smooth muscle (Bulbring, 1961) that the sympathomimetic effects are the result of two opposing actions, one on the cell membrane and thc other on metabolism. G. IONTOPHORETIC APPLICATION OF DRUGS TO NEURONS The blood-brain barrier is circumvented by applying drugs locally

236

E. MARLEY

into the extracellular environment of neurons. The method was introduced by Nastuk (1953), developed by del Castillo and Kata (1955), and successfully adapted for the examination of spinal neurons by Curtis and Eccles (1958a). Synapses occur diffusely over the surfaces of neurons, and presumably chemical substances applied into the extracellular space (Davson and Spaziani, 1959) between the cells would diffuse to some of these synaptic areas. The effects upon the cells are recorded by either extracellular (Curtis and Eccles, 1958a) or intracellular electrodes (Curtis et d.,1959, 1960). When an applied agent has either an excitatory or depressant action on a neuron, this can only be the consequence of the direct interaction between the substance and some neuronal component in the vicinity of the electrode tip. The membrane of neurons consists of electrically excitable membrane and “receptor” membrane which respond to chemical stimuli (Grundfest, 1957). Apart from the indirect action of catecholamines on the central nervous system they may modify neuronal activity by interacting with receptors on the postsynaptic membrane, thereby altering membrane conductance by making certain portions more permeable to particular ions. The sympathomimetic amines may influence the release of transmitter substances from presynaptic terminals or block the postsynaptic action of a transmitter. Noradrenaline and 5-HT applied to neurons in the cat’s reticular formation were ineffective (Curtis and Koizumi, 1961). However, the animals were initially anesthetized with pentothal and by additional anesthetic during the preparation. The results contrasted with those of other experimenters in which recording was taken from reticular units after the intravenous or intraarterial injection of amines. Bonvallet et al. (1956) prepared isolated mesencephalic reticular slabs in which all nervous connections were severed but the circulation was intact. Such reticular slabs contained cells whose discharge was enhanced or inhibited by adrenaline. Bradley and Mollica (1958) recorded the activity of single neurons in the reticular formation. With an intravenous injection of 5-25 pg adrenaline the discharge rate of many neurons increased; that of others slowed and some were unaffected. While response to the intravenous injections often accompanied a rise in blood pressure, similar effects were obtained with adrenaline injected into the carotid artery and preceded changes in systemic blood pressure. Denervation of the carotid sinus did not modify the effects nor could they be elicited from neurons in specific nuclei of the brain stem. The experiments were made in decerebrate cats to eliminate descending corticoreticular influences. The short latency of the effects with intracarotid injections and the fact that they could be obtained after a paralyzing dose of curare argued against a peripheral action of adrenaline.

ADBENERGIC SYSTEM AND SYMPATHOMIMETIC AMINES

237

Bradley and Wolstencroft (1962) applied acetylcholine and noradrenaline iontophoretically to neurons in the lower pons and upper medulla oblongata of decerebrate cats. More neurons were affected by noradrenaline than by acetylcholine, with increase or decrease in discharge rate. Some neurons were affected both by acetylcholine and by noradrenaline, and it is possible that cholinergic and adrenergic receptors were present in the same neurons. Krnjevi6 and Phillis ( 1963a,b) iontophoretically applied amines on single cortical neurons in mammals. There was a tendency toward depression of the activity of most cells, although large doses sometimes caused excitation. The depressant action was usually shown by antagonizing the excitant effect of L-glutamate. Among the most effective compounds were ephedrine, dopamine, and 5-HT; adrenaline, noradrenaline, amphetamine, and histamine were less effective and the most potent amine was less active than GABA.' The action of the catecholamines was quick in onset and rapidly reversible; the principal effect on neuronal firing was a marked reduction in frequency but little depression of spike amplitude. That glutamate firing was depressed suggested that the amines act directly on the postsynaptic membrane. The action of ephedrine was slow in onset, slowly reversible, and spike amplitude rather than frequency was depressed. Dopamine, mescaline, adrenaline, and nor adrenaline applied iontophoretically depressed the orthodromic excitation of neurons in the cat's lateral geniculate nucleus (Curtis and Davis, 1962). The most active depressants of the sympathomimetic amines tested had a terminal primary amino group and either hydroxyl or methoxy groups in the 3,4-position on the benzene ring. Adrenaline, noradrenaline, 5-HTJ histamine (Curtis et al., 1961), dopamine, tryptamine, and a-methyltryptamine (Curtis, 1962) were ineffective on spinal neurons. The negative findings may have been due to the depression of nervous activity in anesthetized animals, particularly by pentobarbitone. Thus intraarterially injected acetylcholine modified the behavior of certain spinal neurons when precautions were taken to prevent afferent impulses from the periphery reaching the neurons (de Molina et. al., 1958); the action of acetylcholine was reduced by anesthesia (chloralose). Tryptamine and a-methyltryptamine facilitate spinal reflexes (Vane et d.,1961); of a number of anesthetics tested (chloralose, halothane, hydroxydione, pentobarbitone) the barbiturate was the only one to seriously depress or abolish the effect of tryptamine on spinal cord reflexes (Marley and Vane, 1963). It is possible that the central effects of many drugs other than tryptamine are vitiated by anesthetics.

' GABA = y-amino-n-butyric acid.

238

E. MARLEY

A defect of the iontophoretic technique is that the amount of drug applied may be inadequate to affect neurons in addition to the difficulty that access to the synaptic membrane may be limited by “synaptic barriers” (Curtis and Eccles, 1958b). Krnjevi6 et d. (1963) tested the effectiveness of drug release from micropipettes by iontophoresis. Release of adrenaline and noradrenaline was the most unsatisfactory ; the transport numbers for different pipettes filled with the same solutions of adrenaline or noradrenaline varied substantially so that a relatively large amount of or practically no drug was released. Large quantities of drug released this way may have nonspecific effects. Iontophoretic studies provide fundamental information of drug action on neurons. However, the selective action of drugs on a functional part of the brain can hardly be established by studying drug action on single cells (Killam, 1962). A change in discharge rate of single neurons may not be very informative; thus the characteristic difference between the neuronal discharge in sleep and wakefulness is not an alteration of average discharge rate but a different pattern of discharge (Creutzfeldt and Jung, 1961).

H. QUANTIFICATION OF THE EFFECTS ON

THE

CENTRAL

NERVOUS SYSTEM

It has proved difficult to delineate the family of pharmacological parameters such as dose-threshold, dose-response, synergism, or antagonism on the central nervous system. Behavior is not readily quantitated, and response, short of intoxication or the production of side effects, is over a restricted dose-range compared with that for in Vitro or even other in vivo systems. Antagonism between drugs acting on the central nervous system is difficult to study; one drug may specifically antagonize another when it is unlikely that they compete for the same central receptors (Gaddum, 1961). Two drugs which stimulate and depress the same part of the central nervous system may specifically antagonize one another although they are not necessarily producing effects a t the same synapse. Interpretation is not made easier by the fact that the brain possesses inhibitory and excitatory neurons, and apparent central depression can arise from stimulation of an inhibitory system or inhibition of an excitatory one. Many methods have been developed since that of Rheinberger and Jasper (1937) for recording cerebral electrical activity and behavior in conscious, unrestrained animals ; the present fashion is for telemetering electrocortical activity using transistorized amplifier transmitters (Vreeland et al., 1958; Fischler et a,?., 1960). Activity, whether electrocortical or behavioral, is more useful if quantitated. A simple way is to rank elec-

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trocortical activity; linear slopes for the plot of rank order or cumulative rank orders against arithmetic or log dose can be obtained (Dewhurst and Marley, 1962). Another way is to measure the duration of some gross change in electrocortical activity, for example, arousal produced by a drug; this requires a preparation with stable sleep electrocortical activity. The sympathomimetic amines tested, such as adrenaline, would necessarily have a short duration of action. Longer arousal would be produced within limits by larger doses of the amine ; with inadequate intervals between doses even of the short-acting amines, the duration of effect may diminish in spite of increased dose (Marley and Key, 1963). Amphetamine would be less suitable to test since tachphylaxis develops (Rothballer, 1957a). Another method is to obtain the thresholds in volts for electrocortical and behavioral arousal on electrical stimulation of the brain-stem reticular formation. Changes in thresholds produced by drugs are plotted against the dose to give dose-response slopes. Incremental doses of amphetamine progressively lowered the threshold for arousal on brain-stem stimulation until eventually a dose was reached a t which the threshold was zero (Bradley and Key, 1958, 1959). Arousal thresholds with auditory or visual stimuli can be obtained if habituation to the stimulus is avoided by positive conditioning (Sharpless and Jasper, 1956). The effects of drugs on conditioning and habituation to arousal stimuli were tested (Key and Bradley, 1960). Amphetamine lowered the threshold for arousal to unconditioned stimuli; it was ineffective on that for conditioned stimuli but restored response to a stimulus which had previously been habituated. Integration is the logical method for quantitating cerebral electrical activity. Riehl e t al. (1960) integrated frequency and voltage changes in electrocortical activity with respect to time; the method relied on visual inspection of the record. Goldstein (1960) used the electronic integrator of Drohocki (1948) ; changes in the rabbit’s cerebral electrical activity produced by morphine and nalorphine were measured (Goldstein and Aldunate, 1960). For the plot of log dose against per cent increase in “electrogenesis” (voltage), linear slopes were obtained with morphine and sigmoid curves with nalorphine. The dc potential recording, although beset with the difficulties that slow potential changes may be due to drifts in base line or artifacts, is a method for recording integrated activity (Eccles, 1961). A simple method for integrating cerebral electrical activity (Dewhurst and Marley, 1964a,b) involves feeding the output from the final stage of the electroencephalograph into an impedance-matching transformer with good low frequency response; the transformer output is rectified and passed into a low inertia dc integrating motor with counter. Large

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potential differences found in normal electrocortical sleep activity or that produced by the catecholamines in the young chicken had large integrals; small potential differences of short duration as in the normal alert electrocorticogram or that produced by amphetamine had small integrals; activity between the two extremes had intermediate integrals. Cerebral electrical activity could be continuously quantitated and it was possible to differentiate drug effects on cerebral electrical activity that would not have been easy by visual inspection of the record. Ideally a number of physiological and behavioral variables should be studied as different measures of activity are often not significantly interrelated (Eayrs, 1954). In the chicken methods were developed for integrating cheeping, motor activity, electromyographic, as well as electrocortical activity (Dewhurst and Marley, 1964a,b). If cumulative integrals were plotted over successive minutes after drug injection, sigmoid or exponential curves were obtained. The total count measured the response to a drug, whereas the curve showed the onset and duration of action. Cumulative integrals were obtained for several doses of the particular amine tested in the same animal. Linear slopes were obtained between log dose and behavioral or the electrical responses. Cheeping, movement, brain, and muscle electrical activity had different thresholds and duration of response to the amines. Linear dose-antagonism slopes could be obtained for the antagonistic action of the catecholamines to the effects produced by the amphetamine-like amines on the four variables. Specific antagonism and tachyphylaxis could also be demonstrated. Analysis of drug effects on behavior by operant-conditioning methods is outside the scope of this review and the interested reader is referred to papers by Brady (1957), Dews (1956), L. Cook and Kelleher (1961), Miller (1957), and Olds et d.(1957).

I. EFFECTS IN GROUPS OF ANIMALS The effects of aggregation are important, for environmental and social stimuli modify drug response (Sabshin and Eisen, 1957; Rathod, 1958). A good deal of information is lost because the drug is usually studied in the isolated animal. The difference in response of single and grouped animals is epitomized in toxicity tests. Thus the LD,, for amphetamine in solitary mice was 90 mg/kg but only 7 mg/kg for groups of mice kept in a confined space (Chance, 1946). Behavior was also modified. Grouped mice proved more susceptible to the excitatory effects of amphetamine and allied drugs than single mice (Gunn and Gurd, 1940). Thus mice given amphetamine evinced little response when kept separately, but if grouped together, abberations of behavior appeared; it seemed as though the activity of

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one mouse excited another. Amphetamine-like amines, unlike other central nervous stimulants, induced in the mouse a state of excitability rather than excitement (Chance, 1946). The result have been confirmed and extended (Lasagna and McCann, 1957; Hohn and Lasagna, 1960). So-called stereotyped behavior was also modified by social stimuli. Selle (1940) described a “stereotyped” response to amphetamine in young chickens. Within a few minutes of injection the neck and wings extended and there was increased motor activity. At first the chicken ran round aimlessly but ataxia ultimately developed and the chick remained stationary for long periods, uttering a continuous high-pitched twittering. A similar picture was elicited by (+)-deoxyephedrine, P-phenyl-a-aminobutane, and P-phenyl-tert-butylamine (Clymer and Seifter, 1947) ; phenmetrazine, tuaminoheptane, and methyl phenidate (Key and Marley, 1962) ; and cyclopentamine and a-methyltryptamine (Dewhurst and Marley, 1964a). Whereas amphetamine readily evoked electrocortical alerting, increased electromyographic activity, and postural changes whatever the circumstances of the bird, the stereotyped twittering only occasionally occurred in isolated birds. If the bird was handled or other birds were present, twittering developed. In a cross-over test there was a significantly greater incidence of amphetamine-induced twittering in accompanied (17/18) than in isolated birds (2/18) (Dewhurst and Marley, 1964a).

J. SIDE EFFECTS IN MAN Side effects with moderate doses of amphetamine-like amines are usually minimal and consist of anorexia, insomnia, etc. Acute or acuteon-chronic intoxication may develop after the ingestion of large amounts and the side effects emulate the transient changes elicited in animals by comparatively much smaller intravenous doses of the drugs. Few signs are supposed to be present with amphetamine intoxication but there may be a coterie of signs including mydriasis, impaired pupillary response to light and accommodation, tremor of the tongue and limbs, augmented tendon jerks with increased limb tone, and facial tics and twitching (Marley, 1960b). Mydriasis with amphetamine intoxication (Shorvon, 1945; Herman and Nagler, 1954; Patuck, 1956; Connell, 1958) appears from experiments in the cat endphale isold preparation to be due to a combination of central inhibition of parasympathetic pupil constrictor tone and a direct action of amphetamine on sympathetic postganglionic nerve endings in the iris (Marley, 1961b). Tremor in man after amphetamine was mentioned by Monroe and Drell (1947) and Knapp (1952) and in animals by Alles (1927). Mydriasis, tremor, and restlessness occur after overdose with

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ephedrine (Goodman and Gilman, 1955), and the drug produces a picture not a t all unlike amphetamine intoxication (Locket, 1957). Similar features occur with phenmetrazine intoxication (Bartholomew and Marley, 1959). Restlessness is marked during intoxication ; increased abnormal motor activity was found after pipadrol (Fullerton, 1956). In mice, psychomotor stimulants such as methylamphetamine and cocaine greatly increase coordinated activity, while central stimulants such as picrotoxin are ineffective or depress activity (Dews, 1953). Small ticlike movements of the hands, feet, eyes, ears, and especially of the lips and mouth with frequent touching of the nasolabial region have been produced in monkeys with amphetamine or methyl phenidate (Cole and Glees, 1957), mescaline, and other phenylethylamine (Kluver, 1958). Ataxic signs including nystagmus are rare with intoxication by amphetamine-like amines but frequent with intoxications produced by central depressants (Marley, 1960b). The difference may be because impairment of postural mechanisms is produced by central depressant drugs (Hondelink, 1932; Beecher et al., 1939), whereas the amphetaminelike amines facilitate postural activity. Thus righting and other postural activity returns in decerebrate cats after (+)-amphetamine (Maling and Acheson, 1946, Macht, 1950), after pipadrol (B. B. Brown and Lerner, 1954), and in decapitate dogs after ephedrine (Hinsey et al., 1931). IX. Blockade of Adrenergic Neuron and Receptor

Adrenergic antagonists, if sufficiently specific and if able to differentiate exquisitely between the direct and indirect actions of sympathomimetic amines, would be of inestimable value in defining the physicochemical properties of the adrenergic receptor and in classifying the sympathomimetic amines. A large number of adrenergic-blocking drugs are now available; rather than consider the entire gamut, a paradigm for each category will be discussed. A. BLOCKADE OF ADRENERGIC RECEPTOR Analysis of the structural isosterism relating the Dibenamine ethylenimonium ion with P-phenylethylamine made it certain that the noncompetitive block produced by Dibenamine and its congeners was ascribable to a chemical interaction with the same receptor sites that bind agonist amines (Belleau, 1958, 1959a,b). There were two types of evidence: (1) All active adrenergic-blocking agents obeyed the “phenylethylamine pattern rule” which provides for the interchangeability in a drug of ammonium ions and partial carbonium ions as far as interactions with anionic groups are concerned. Thus, an

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ethylenimonium ion could initially interact electrostatically with an anionic site (Belleau, 1958), followed by a rearrangement bringing an electrophilic carbon sufficiently close to the anionic site to allow esterification (or alkylation) of the latter. (2) The influence of substances on the aromatic ring of the adrenergic-blocking agents paralleled the effects of the same substituents on the excitatory activity of sympathomimetic amines. Nickerson and Nomaguchi (1953) examined the effect of sympathomimetic amines on the blood pressure and the chronically denervated nictitating membrane in cats treated with Dibenamine. Excitatory responses (effect on a-receptors) of the denervated nictitating membrane to all sympathomimetic amines tested were abolished. The pressor action was lost but depending on the structure of the amine a hypotensive effect emerged. Four structural features were of importance for this: (1) phenolic hydroxyl groups (especially in the 3,4-position), (2) N-alkyl substituents, (3) a-aliphatic substituents, and (4) the hydroxyl on the /3-carbon. The findings are reminiscent of the configuration of molecules with inhibitory properties found by Lands and Tainter (1953). Significant hypotensive action was not found unless the amine had a t least one phenolic hydroxyl ; dimethoxyphenyl, piperonyl, or 2,3dihydroxyphenyl substituents were ineffective as were the aliphatic and imidaeoline amines. N-Alkyl substitution did not provide significant inhibitory activity in the absence of phenolic hydroxyls, as noted by Barger and Dale (1910). A feature not emphasized in other structureactivity studies was the importance of the a-aliphatic substituent for inhibitory activity although the series phenylethyl-, phenylisopropyl-, and phenyl-2-butylamine was associated with increasing ability to relax the rabbit jejunum (Marsh, 1948). As with N-alkyl substituents, aalkyl substituents were much less effective in inducing depressor activity in the absence of phenolic hydroxyls. The combination of an N-methyl and an a-methyl provided marked inhibitory properties in the presence of only one phenolic hydroxyl. Although all adrenergic-blocking agents influence the central nervous system (Nickerson, 1959) their blocking action against the central effect of sympathomimetic amines is uncertain. This is not because of difficulty in penetrating the central nervous system as both Dibenamine and Dibenzyline have high lipid solubility a t body pH. Thus the Dibenamine type of drug prevents neither the hyperventilation produced by adrenaline (Nickerson and Goodman, 1947) nor the increased motor activity in mice induced by certain sympathomimetic amines (Tripod, 1952). It may be that the receptors in the brain for sympathomimetic amines differ in some subtle way from the a- and /3-receptors in peripheral tis-

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sues, Phentolamine which blocks the peripheral action of adrenaline also suppresses the electrocortical alerting which would otherwise occur (Capon, 1960) ; this could be interpreted that alerting produced by adrenaline is a consequence of its peripheral action rather than that phentolamine blocks adrenaline receptors in the central nervous system. If it is the unsubstituted cationic head of the sympathomimetic amine which is important for excitatory activity, then presumably amines with this side-chain termination but different ring structure (tryptamine, histamine) will act at the same anionic site. Consequently, adrenergic, histamine, and tryptamine antagonists would have reciprocal blocking capacity with greater antagonistic potency to one or more of the agonists than to all. This is in fact so and any arylhaloalkylamine exhibiting adrenergicblocking properties is capable of antagonizing various responses to histamine (Nickerson, 1949). Dibenamine itself has little antihistaminic activity but certain of its congeners, particularly those with l-naphthylmethyl and phenoxyethyl substituents, are potent antihistamines ; Dibenzyline is quite active in this regard. The blockade of responses to histamine appears to be characterized by the same persistence and stability as the blockade of adrenergic stimuli. Indeed the a-naphthylmethyl derivatives of /3-chloroethylamine antagonize histamine as strongly as they do adrenaline (Loew, 1950). Stone and Loew (1952) studied the antagonism of several arylhaloalkylamines to adrenaline, noradrenaline, and histamine on the guinea pig-isolated seminal vesicle. The antagonism to histamine bore no relation to that of adrenaline; likewise antihistamine potency did not parallel the anti-acetylcholine effects. Stone and Loew interpreted this as contradicting (‘the oft-repeated concept that histamine and adrenaline, and histamine and acetylcholine act upon the same or closely related receptors.” This lack of specificity extends to other adrenergicblocking substances. Piperoxan, phentolamine, and dihydroergotamine reduced the relaxant response to adrenaline on the guinea pig stomach; the contractor responses to histamine and acetylcholine were similarly reduced. The lack of specificity of these drugs, in the strong concentration needed to reduce the relaxant effect of adrenaline, detracted from the significance of the results, although they were compatible with a true antagonism to sympathin released from adrenergic nerve endings in the wall of the stomach (Paton and Vane, 1963). Phenoxybenzamine, previously thought to block specifically a-adrenergic responses (Furchgott, 1954), was extremely potent against the excitatory actions of acetylcholine and of cholinergic nerve stimulation; it was often more effective

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against these than against adrenergic nerve excitation (Boyd et al., 1963). The matter is further complicated by the fact that when the sympathomimetic action of amphetamine-like amines is absent an underlying action on tryptamine receptors is revealed (Vane, 1961). The tryptamine activity can be abolished by bromolysergic acid or methysergide, which interacts either with the ring or the anionic site, and by phenoxybenzamine, which competes for the anionic receptor. Perhaps a more rational classification of antagonists would be to divide them into those competing with the cationic head of the agonist for the anionic site, and those blocking the interaction of the &carbon and phenolic hydroxyls with their respective receptors. Dibenamine and phenoxybenzamine belong to the first category; antagonists which by virtue of a substituted cationic head do not readily interact with the anionic receptor belong to the second category and would include phenylisobutylnoradrenaline and the @-blockingsubstances DCI and pronethalol The classification would account for the Dibenamine blockade of amines with similar cationic heads such as histamine, noradrenaline, and tryptamine; it would also account for the appearance of inhibitory properties by molecules with phenolic hydroxyls after Dibenamine blockade. Compounds such as dihydroergotamine would have two antagonistic actions, one due to interaction with the anionic site and the other attributable to molecular bulk hindering access to the receptor by amines with phenolic hydroxyls. B. BLOCKADE OF ADRENERGIC NEURON The development of adrenergic nerve-blocking agents is relatively recent. The most noteworthy are bretylium and guanethidine; as there are striking similarities between the two, both will be considered. Bretylium, a quaternary benzylammonium salt, interferes with adrenergic transmitter release by depressing the excitability of adrenergic nerve terminals without affecting the reactivity of chromaffin tissue (Boura and Green, 1959). The blockade by guanethedine {[2(octahydro-l-azocinyl) ethyl J guanidine sulfate} was described by Maxwell et d.(1960). Both drugs have an initial sympathomimetic action which persists after bilateral adrenalectomy (Boura and Green, 1959; Gokhale et al., 1963) and is due to release of tissue catecholamines. The sympathomimetic effect is reduced or abolished by prior reserpinization (Bein, 1961; Gillis, 1961; A. F. Green, 1961), depressed by the adrenergic receptorblocking agents Dibenzyline (A. F. Green, 1962) or phentolamine (Max-

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well, cited by Richardson and Wyso, 1960), and potentiated by pyrogallol which inhibits O-methyltramferase. Adrenergic neuron blocking next develops so that response to postganglionic sympathetic nerve excitation dwindles, whereas that to injected adrenaline or noradrenaline remains (Boura and Green, 1959; Maxwell et al., 1960; A. F. Green, 1962). Both substances produce transient ganglionic block (Boura and Green, 1959; Bein, 1961). According to Abercrombie and Davies (1963) guanethidine appears to interfere with the synthesis of transmitter. Certainly both drugs rapidly abolish the liberation of Hs-noradrenaline into the splenic venous blood on nerve excitation, a t a time when the tissue concentration of Hs-noradrenaline is still high (Hertting et al., 1962). The nerve blocking action was reversed by dopamine and cocaine (Boura and Green, 1959; Day, 1962; A. F. Green, 1962). The blockade was reversed also by (+)-amphetamine, mephentermine, hydroxyamphetamine, ephedrine, and phenylethylamine ; if these amines were given first, then the adrenergic neuron-blocking agents were ineffective (Day, 1962). Guanethidine and amphetamine appear to act on the same receptor (Day and Rand, 1963), and the antagonism by (+)-amphetamine of the sympathetic nerve block by guanethidine fulfilled the conditions of competitive antagonism defined by Arunlakshana and Schild (1959). As a consequence of nerve blocking there is a gradual depletion of tissue noradrenaline (A, F. Green, 1962; Sheppard and Zimmerman, 1959; Cass et al., 1960; Cass and Spriggs, 1961). Bretylium accumulates in sympathetic ganglia and their postganglionic nerve trunks at concentrations far exceeding those in other tissues (Boura et al., 1960). There is a rough correlation between the degree of accumulation and the tissue concentration of dopamine; bretylium may have a high affinity for sites associated with dopamine (A. F. Green, 1962). Whether guanethidine concentrates in the adrenergic system is not known. Because tissue stores of noradrenaline are only gradually depleted it could be surmised that with “acute” bretylium or guanethidine administration, and in spite of the absence of response to nerve excitation, indirect-acting sympathomimetic amines would still be active. With “chronic” administration, and as a consequence of nerve block, the tissue noradrenaline dwindles with consequent ineffectiveness of the indirectacting amines. With the onset of bretylium blockade the effects of intravenous adrenaline and noradrenaline were augmented. The amines elicited greater pressor responses, larger contractions of the nictitating membrane, and profounder vasoconstriction in perfused rabbit ears (Boura and Green, 1959). The effect of intravenous tyramine, amphetamine, methylam-

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phetamine, and ephedrine were also greater after bretylium in the acute experiment (A. F. Green, 1961; Hukovi6, 1960). I n cats, in which mydriasis on excitation of the cervical sympathetic nerve was abolished by bretylium, the iris was sensitized to sympathomimetic amines (Marley, 1962) ; the threshold mydriatic dose for Epinine and tyramine was lowered and the mydriatic action of amphetamine prolonged. I n animals treated with guanethidine for 2 days, amphetamine and ephedrine produced their usual rise in blood pressure, although adrenergic nerves were blocked (Zaimis, 1960). The response to sympathomimetic amines differed after chronic administration of bretylium. In cats given daily large doses of bretylium for several months the sensitivity of the nictitating membrane to injected adrenaline or noradrenaline increased progressively for about 2 weeks (A. F. Green, 1961); thereafter, the response dwindled. The effect of tyramine was initially enhanced but declined at 4 weeks and was virtually abolished a t 6 months. The effects of ephedrine and methylamphetarnine were also reduced after several weeks, These changes were analogous with those produced by sympathetic postganglionic nerve degeneration or reserpine and could be related to the gradual depletion of catecholamine content of nerves (A. F. Green, 1961). Maxwell et al. (1960) examined the pressor effects of various sympathomimetic amines in dogs 48 hours after the injection of guanethidine. The amines could be divided into three groups. I n group 1, the pressor responses to amines with phenolic hydroxyls in the 3,4-positions (noradrenaline, adrenaline, Cobefrin, and Epinine) were significantly augmented; responses to dopamine, phenylephrine, and oxedrine were not enhanced. I n group 2, the effect of amines with a hydroxyl on the p carbon (ephedrine, phenylpropanolamine) were partially suppressed, whereas the actions of group 3 amines, those without hydroxyls or with a phenolic hydroxyl in the 4-position (amphetamine, phenylethylamine, methylamphetamine, and pholedrine) ,were abolished. A first glance, as with blockade of adrenergic receptors, provides a deliciously simple but fallacious impression. Thus, the lack of specificity already noted for receptor blockade applies to drugs blocking adrenergic neurons (Boyd et al., 1963). Bretylium antagonired the effect of acetylcholine and of nerve excitation in cholinergically innervated preparations. Many adrenergic blocking agents are anti-cholinesterases (Boyd e t al., 1960). Adrenergic and cholinergic blocking may not be truly distinguishable; drugs a t one end of the spectrum principally block cholinergic nerves, those a t the other end principally adrenergic nerves (Boyd et d., 1963). Boyd et al. (1963) concluded that “the use of autonomic drugs

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as analytical tools was not reliable unless combined with histochemical examination of the preparation and assay of substances released upon nerve stimulation.” X. Inactivation of Amines

There are a number of routes for the inactivation of sympathomimetic amines. The subject has been reviewed by Axelrod (1959) and Iisalo (1962). A. DEAMINATION The enzyme monoamine oxidase occur8 in most body tissues (Blaschko, 1952) and it deaminates alkyl and aromatic amines to the corresponding aldehydes. It is easy to show in vitro that the enzyme oxidizes the side chain of amines such as adrenaline, noradrenaline, tyramine, phenylethylamine, tryptamine, and 5-HT. The evaluation of monoamine oxidase in the metabolism of phenylethylamines in wivo received impetus from the use of amine oxidase inhibitors. The role of the enzyme was more limited than had appeared from in vitro studies. Phenylethylamine and tyramine were deaminated by amine oxidase, but not phenylethylamines in which the amino group was attached to other than the terminal carbon atom; adrenaline, noradrenaline, and 5-HT were not potentiated by amine oxidase inhibitors (Griesemer et al., 1953; Corne and Graham, 1957; Vane, 1959). The role of brain amine oxidase is far from clear; although the amine oxidase activity of some parts of the brain is high compared with that of other tissues, that of another inactivating enzyme, catechol-0-methyltransferase, is highest in the neurohypophysis. However, in spite of the importance in some species of the 0-methylation of noradrenaline and adrenaline in vivo (Axelrod, 1957; Armstrong et al., 1957) monoamine oxidase appears t o be implicated in the metabolism of these amines. For instance, the catecholamine content of heart muscle is increased after iproniazid administration (Pekkarinen et d.,1958; Pletscher, 1958) ; a corresponding increase also takes place in the brain after injection of several amine oxidase inhibitors of the hydrasine or non-hydrazine type (Shore et al., 1957; Finger, 1960; H. Green and Erickson, 1960). The plasma adrenaline content is increased by harmaline and iproniazid (Eakins and Lockett, 1961). I n cats pretreated with phenylisopropylhydrazine the effects of 5-HT injected into the lateral cerebral ventricle were greatly intensified and prolonged; the effects of adrenaline were potentiated to a lesser extent (Schain, 1961). Those of noradrensline, dopamine, and tryptamine were not intensified and were prolonged only to a slight degree. The main function of mono-

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amine oxidase under physiological conditions, a t least in the brain, is the intracellular regulation of noradrenaline and 5HT within neurons, so that constant access of these amines to receptors is interrupted (Spector et al., 1960a). A large proportion of dopamine is destroyed by amine oxidase (Blaschko, 1956). The relative dependence of the different amines on monoamine oxidase requires simultaneous measurement of all the substrates. Dewhurst (1961) measured tryptamine, 5-HT, metanephrine, and normetanephrine excretion in man (8 adults) before and 2 weeks after phenelzine administration. All substrates were excreted in significantly larger amounts; tryptamine excretion increased fourfold, 5-HT threefold, and the metanephrines twofold. VMA, the main product of monoamine oxidase action on the catecholamines, was only reduced by 30% (Dewhurst, 1961) in contrast to the gross reduction of 5-hydroxyindoleacetic acid excretion (Dewhurst and Pare, 1961). An enzyme in liver microsomes can deaminate amphetamine to phenylacetic acid and ammonia in the presence of reduced triphosphopyridine nucleotide (TPNH) and oxygen (Axelrod, 1955). The enzyme differs from other deaminating enzymes such as amine oxidase, Land D-amino acid oxidase, and glutamic acid dehydrogenase with respect to its substrate specificity, intracellular localization, and cofactor requirements (Axelrod, 1955). Amines having a phenylisopropylamine or phenylbutylamine structure were extensively transformed by the enzyme, while phenolic-substituted amines, phenylethylamines, and aliphatic amines were metabolized slightly or not a t all (Axelrod, 1955). I n man and dog, although a major proportion of mescaline is excreted unchanged (Cochin et al., 1961; Sarkar et aZ., 1957) i t is metabolized by diamine rather than monoamine oxidase (Zeller et d., 1958). Beyer (1941) observed that ascorbic acid can deaminate a number of phenylethylamines and phenylisopropylamines nonenzymically and he has confirmed this in vivo (Beyer, 1942).

B. O-METHYLATION O-Methylation has been proposed as the principal pathway in the metabolism of catecholamines. Catechol-O-methyltransferase is the enzyme primarily concerned in the inactivation of circulating and possible locally released catecholamines (Axelrod, 1961). Catechol-O-methyltransferase and monoamine oxidase may differ in that the transferase inactivates circulating adrenaline and noradrenaline, whereas monoamine oxidase metabolizes tissue catecholamines (Spector et d., 1960a)b). I n man, approximately 68% of administered adrenaline is O-methylated to metanephrine and 23% is deaminated, oxidized, or

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reduced, most of this then being O-methylated; the remainder is excreted as unchanged and conjugated catecholamines. Noradrenaline would appear to be similarly dealt with. There are species differences; in the rat and in other species, but not in man, part of the metanephrine formed is O-demethylated to adrenaline and remethylated again (Axelrod and Szara, 1958). Metanephrine and normetanephrine possess weak physiological properties (Evarts et al., 1958; Key and Marley, 1962; Marley, 1962) suggesting that O-methylation is an inactivation process. O-Methylation and also the methylation of noradrenaline to adrenaline, are dependent on the presence of the coenzyme, S-adenosyl methionine, as a source of methyl groups. The brain is able to synthesize this substance from ATP and methionine (Axelrod et al., 1959) and is thus fitted to O-methylate catecholamines. Catechol-O-methyltransferase is widely distributed among a variety of tissues and species and is localized in the soluble fraction of the cell (Axelrod and Tomchick, 1958); of all the tissues examined, enzyme activity was consistently highest in the liver. The presence of the enzyme in organs on which adrenaline and noradrenaline exert their effects suggests that i t might act locally in the transformation of these substances. When the catecholamines are released they would first appear in the cell sap and be exposed to catechol-0-methyltransferase ; in order to be attacked by amine oxidase they must penetrate the mitochondria. The methylated compounds normetanephrine and metanephrine are either conjugated with glucuronic acid or deaminated by monoamine oxidase (Axelrod et ul., 1958) t o form 3-methoxy-4-hydroxymandelic acid. Although catechol-0-methyltransferase is chiefly concerned with adrenaline and noradrenaline metabolism, i t does not necessarily follow that it terminates the action of these hormones. Other mechanisms such as tissue binding are important.

C. N-DEMETHYLATION In the dog and guinea pig the principal metabolic pathway for ephedrine involves rapid N-demethylation to the relatively stable norephedrine, and it is probable that the activity of ephedrine in mediated through this metabolite. Norephedrine is excreted mainly unchanged in the urine (Axelrod, 1953). There are species differences in the metabolism of ephedrine. The rat demethylates ephedrine slowly, and considerable amounts of the substances are excreted unchanged and as hydroxylated derivatives. About half of administered methylamphetamine is demethylated to amphetamine in the dog; pharmacological activity of methylamphetamine appears to be mediated through its metabolite.

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D. O-DEMETHYLATION A minor metabolite found in the urine in man after mescaline ingestion is a conjugate of 3,4-dihydroxy-5-methoxyphenylaceticacid. E. HYDROXYLATION The main metabolic route of (+)-amphetamine in the dog involves hydroxylation to p-hydroxyamphetamine ; after the administration of (-)-amphetamine only small amounts of p-hydroxyamphetamine are excreted. The difference in the metabolism of these amphetamine isomers is presumably due to a stereospecific enzyme (Axelrod, 1955) which deaminates the (-) - more readily than the (+)-isomer. When hydroxyamphetamine is administered it appears in the urine partly free and partly conjugated.

F. BINDINGOF AMINES There is an excellent review of the binding of amines by J. P. Green (1962). Several hours after adrenaline or noradrenaline injection (Axelrod et al., 1959; Axelrod and Tomchick, 1960) and long after their obvious pharmacological effects have abated (Wylie et al., 1960) the major portion of the amine can be recovered unchanged from tissues. Tissue binding thus prevents enzymic alteration of the amines, and binding of catecholamines to other than their specific receptors may be a means of diminishing their effects. Consequently, blockade of these alternative binding sites should allow larger amounts of the amines to act on specific receptors and, by mass action laws, potentiate their effects. Dibenzyline reduced noradrenaline uptake by tissues (Hertting et al., 1961d) which may explain how i t potentiates the effect of catecholamines (Holzbauer and Vogt, 1955). Other substances potentiating the effect of noradrenaline and inhibiting its uptake by tissues are bretylium, chlorpromazine, ergotamine, guanethidine, imipramine, and mescaline (Dengler et al., 1961b). Cocaine and denervation may block amine uptake (Trendlenburg, 1959; Hertting et al., 19614. Blockade of binding sites should accelerate amine metabolism because of the accessibility of the amines to inactivating enzymes. Implicit in these assumptions is that there is competition between the adrenergic receptor and alternative sites for the binding of amines. At physiological pH, amines are mostly in the cationic form (G. P. Lewis, 1954) and therefore capable of forming salt linkages with anionic groups, such as carboxylate, sulfonate, and phosphate. Hydrogen bonding of the primary amino group with the same anion doubles the strength

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of the ionic bond. Hydrogen bonding may also occur with the phenolic] alcoholic, or catecholic groups. These bonds probably exercise a major function in the binding of amines to tissue components. With all the amines, van der Waal’s forces supplement the strength of bonding, especially where complementary exists between the amine and the substance with which it interacts. The strength of the van der Waal’s bond is about 0.5 kcal, that of the hydrogen bond 2-5 kcal, that of the ionic bond 5 kcal, and for the reinforced ionic bond, 10 kcal (Albert, 1960). Amines in the adrenal medullary granules may be bound to protein, which is held in contracted state by hydrogen bonds (Hillarp, 1961). Amines may also be fixed in covalent linkage to proteins (Waelsch, 1961) ; there was little specificity as to the amine that was fixed, ammonia and glycinamide being incorporated as well as amines of particular importance to the central nervous system, such as histamine, noradrenaline, 5-HT, and mescaline. XI. Conclusion

The molecular pedigree of the sympathomimetic amines ramifies fascinatingly back to adrenaline. Inevitably a brief acquaintance with any of the amines implies ultimately a long tryst with the autonomic and central nervous systems. Some facets of these interrelations have been discussed. The approach has been dogmatic and consequently requires qualification. The sympathomimetic amines have been classified in three “ideal” groups, although in fact there is a graded transition in activity through members of one group to those of the next. A wealth of problems require clarification. These include the extent to which cholinergic fibers are implicated in noradrenaline liberation by sympathetic postganglionic nerves, the number and size of the components of the noradrenaline store, elucidation of antagonism to the peripheral and central action of sympathomimetic amines, and, not least, the function of amines in, and their effect on, the central nervous system. Clues to these problems abound but their nomination is as yet imprecise. ACKNOWLEWMENTH The work of the author waa supported by grants from the Bethlem Royal and Maudsley Hospitals Research Fund, by the Central Research Fund of London University, by the Ford Foundation, and by the Medical Research Council, England. Much helpful discuasion with Dr. W. G. Dewhurst is gratefully acknowledged.

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Pharmacological Aspects of Drug Dependence G. A. DENEAU AND M. H. SEEVERS Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan

I. Introduction . . . . . . . . . . . . 11. Tolerance . . . . . . . . . . . . . 111. Physical Dependence . . . . . . . . . . IV. Psychogenic Dependence . . . . . . . . . V. Attempts to Find Nondependence-Producing Analgesics . A. Compounds with Morphine-Like Action . . . . . B. Structure-Activity Relations (SAR) . . . . . . C . The Narcotic Antagonists . . . . . . . . VI. Antitwives . . . . . . . . . . . . References . . . . . . . . . . . .

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1. Introduction

I n the interest of more accurate scientific and medical reporting, the Expert Committee on Drugs Liable to Produce Addiction of the World Health Organization has recently abandoned the terms habituation and addiction in favor of the general designation drug dependence. In accord with this action, those biological responses which follow chronic intoxication with psychotropic drugs-tolerance, physical dependence, and psychogenic or psychic dependence-are used herein. II. Tolerance

Tolerance to the effects of drugs occurs in many organisms from bacteria to man. Mammals develop tolerance to many types of drugs which affect the nervous system and the vascular system. Several monographs, reviews, and symposia have dealt extensively with the various forms of tolerance in recent years (Krueger et al., 1941; Isbell and Fraser, 1950; Seevers and Woods, 1953; Eddy, 1955; Reynolds and Randall, 1957; Butler, 1958; Way and Adler, 1960; Seevers and Deneau, 1963, 1964). Drug dependence in man does not develop to many of the classes of drugs which induce tolerance (e.g., atropine, organic nitrates), and tolerance does not develop to some of the drugs to which man may become dependent (e.g., cocaine). Both tolerance and dependence develop to two major classes of central nervous system depressants, narcotic analgesics and the sedative hypnotics. A subject who is tolerant to a drug is also tolerant to the other drugs within the same pharmacological class, but specific cross tolerance does not occur between the different classes of 267

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drugs. Hypotheses concerning the possible mechanisms of tolerance development and the many works designed to test these hypotheses have been thoroughly discussed in the reviews listed above. At present, only two mechanisms, increased detoxication and cellular tolerance, remain in consideration. Way and Adler (1960) have recently reviewed the extensive studies concerning the detoxication of the narcotic analgesics and have concluded that any alterations which have been observed are disproportionately small in comparison to the extent of tolerance developed to morphine. Thus, by eliminating the other suggested explanations for the development of tolerance, the only reasonable explanation of morphine tolerance that remains is decreased cellular sensitivity of the central nervous system. Axelrod (1956) has offered a suggestion as to how decreased cellular sensitivity might occur. He found that the ability of liver microsomes, obtained from morphine-tolerant rats, to demethylate several narcotic analgesics in vitro was markedly reduced. Reasoning that the receptors for morphine in the liver were probably similar to the morphine receptors in the central nervous system, he suggests that a similar reduction in the number of receptors might occur in both organs. If the number of receptors in the central nervous system is decreased, the administration of a given dose of morphine would result in fewer drug-receptor combinations and consequently a decreased effect. Way and Adler (1960) have cited many objections to this concept, but a determination of its validity cannot be made until the morphine receptors in the central nervous system are identified and quantitatively measured. Takemori (1961) has demonstrated that morphine, barbiturates, and alcohol depress the potassium-induced stimulation of respiration of raf brain cortex slices. If the rats are previously made tolerant to morphine, however, morphine no longer exhibits this effect in vitro although the barbiturates and ethanol produce the same effects as in normal rats. Although research on the mechanisms of tolerance to the sedative hypnotic drugs has been less extensive than with the narcotic analgesics, certain conclusions seem to be warranted, Increased rates of detoxication have been demonstrated clearly, and some degree of central nervous system tolerance may also occur. The activity of enzymes residing in the liver microsomes, which metabolize barbiturates and other types of drugs, has been shown to be stimulated by pretreatment with a variety of drugs. Most of the work on enzyme activation has been conducted by two groups of investigators, both of whom have recently reviewed their work (Connay and Burns, 1962; Remmer, 1962). Increased enzymic activity becomes apparent 12 to 24 hours after administration of the priming dose,

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is maximal a t 72 hours, and diminishes gradually thereafter. Tolerance, as evidenced by decreased sleeping time to a test dose of barbiturate, develops and disappears in a time sequence which corresponds exactly to the increased oxidative enzyme activity of the liver microsomes. The increase in activity may be induced not only by such various drugs as chlorcyclizine, phenobarbital, phenylbutazone, orphenadrine, and aminopyrine, but also by drugs such as SKF 525A, which initially inhibit a variety of drug-metabolizing enzymes. Remmer (1962)noticed that some sedatives were more effective than others in the induction of increased enzyme activity; he also found that ethanol had no effect in this regard. Remmer also pointed out that oxidation of barbiturates in man is much less pronounced than in rats and that a cellular adaptation of the central nervous system is probably partially responsible for the tolerance to sedatives in man. Unfortunately the extent of species variation in cellular and metabolic mechanisms of tolerance is not clearly understood. Most of the experiments on metabolic tolerance have been conducted with rats and rabbits, but a few have been done with dogs. In rats, the induced increase in enzymic activity disappears in 28 days, whereas this effect persists for a t least 4 months in dogs. To what extent does induced enzyme activity contribute to tolerance to sedatives in man? And how long does such a mechanism, if it exists, persist? The lack of effect of ethanol as a stimulus for enzyme induction in rats (Remmer, 1962) is clearly a t variance with results in man. Isbell e t al. (1955) have demonstrated a metabolic tolerance to ethanol in some patients. Since the groundwork for the phenomenon of metabolic tolerance has been delineated so clearly in rodents, it is to be hoped that these studies will be extended to the higher species, 111. Physical Dependence

Isbell and Fraser (1950) have reviewed physical dependence and have emphasized that two different types of this phenomenon are known. One type is that which results from chronic administration of the narcotic analgesics; the other type may be induced by the sedative-hypnotic class of drugs. I n both cases, when drug administration is abruptly discontinued after a prolonged period of chronic treatment with large doses, syndromes indicative of gross physiological imbalance occur. The syndrome that is related to the narcotic analgesics is characterized by signs of psychic and somatic hyperirritability as well as hyperirritability of both divisions of the autonomic nervous system. The syndrome that follows withdrawal of the sedative hypnotics is characterized by psychoses and grand ma1 seizures.

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The clinical techniques for evaluating a drug’s capacity to induce physical dependence have been discussed by Isbell and Fraser (1950). In essence these techniques are: (1) The development of physical dependence to an agent as the rcsult of chronic, cohtinuous administration of the drug for several weeks. Determination of the existence and extent of physical dependence depends upon the appearance of a characteristic abstinence syndrome when drug administration is abruptly discontinued. The existence of physical dependence cannot be established while the drug is being administered in adequate quahtitiks. If one of the narcotic analgesic antagonists (e.g., nalorphine, levallorphan) is administered, however, an “acute abstinence syndrome” is precipitated but the signs persist only as long as effective concentrations of the antagonist remain in the organism. The onset, intensity, and duration of the abstinence syndrome which results on abrupt withdrawal determine the degree of the drug’s physical dependence capacity. (2) The test drug is substituted for a control drug (e.g., morphine) in an animal or an individual known to be physically dependent on morphine for a period in excess of the normal duration of the morphine abstinence syndrome. If abrupt discontinuance of all treatment now results in the appearance of an abstinence syndrome, the test drug is stated to be morphine-like since i t sustains the state of physical dependence. (3) If a test drug is administered during the early stages of the morphine abstinence syndrome and causes a reversal in the course of the syndrome as it increases in intensity by specifically suppressing all of the morphine abstinence signs, then the test drug is also stated to be morphine-like and to possess the capacity to suppress abstinence signs for a period corresponding to the duration of the effect of the test drug. It should be emphasized that the nonspecific suppression of only one or two abstinence signs (e.g., the suppression of vomiting by an antiemetic such as phenothiaeine) does not of itself constitute evidence that the drug is morphine-like. The factors which influence the development of physical dependence and the poitulates concerning the mechanism underlying its development have recently been reviewed (Seevers and Deneau, 1963, 1964). The factors which favor the optimal development of physical dependence to the narcotic analgesics are related to the nature of the particular drug, to’the manner of its administration, and to the species. . Factors related to the nature of the drug: (1) The drug should produce depression of a wide variety of nervous functions. (2) The drug

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should possess a wide effective dose range before signs of toxicity such as convulsions supervene. With such a drug the dosage can be increased greatly as tolerance develops during chronic administration. (3) The drug should possess a fairly long duration of action so that continuous tissue saturation is more easily maintained. Factors related t o the administration of the drug: (1) Certain finite dosage levels must be maintained before significant abstinence signs are observed upon withdrawal. The degree of physical dependence is related to dose almost linearly to a point which depends upon the drug and the species. Beyond this point the degree of dependence does not increase significantly as the dose is increased. (2) The daily dose should be divided and administered at such intervals that the organism is continuously exposed to the drug. Frequency of administration is conditioned by the total duration of action of the test drug. I n order to assure adequate and continuous drug effects, the dose response curves should overlap. (3) Continuous administration of a narcotic for a t least several days is required before abstinence signs can be observed on abrupt withdrawal of treatment. The precise minimum period of treatment is not known, but it is shortest when the properties of the drug in question approach those listed above and when the highest tolerated dose is administered. On a stable dose of 3 mg/kg morphine sulfate every 6 hours to the monkey (Macaca mu2atta) maximum dependence does not develop for 2 months, although it is nearly complete a t the end of 1 month. Factors related to animal species: No comprehensive study which compares the various species with respect to ease of development of physical dependence is known. Based on the per kilogram dose required to produce definite dependence to morphine, man appears to be the most sensitive species. The monkey (Macaca mulatta) and dog both develop high-grade physical dependence to morphine. The constipating and emetic effects are so severe in some dogs, however, that nutritional problems interfere with and may even preclude the development of dependence. The signs of abstinence are much more erratic in the dog than in the monkey. The rat develops physical dependence to morphine but a higher dosage is required than for other species and the abstinence signs are less distinct. Cats, rabbits, guinea pigs, and mice develop only ill-defined, if any, physical dependence to morphine. The factors which regulate the extent of development of physical dependence to the sedative hypnotics have not been studied extensively. The studies of Fraser et a,?. (1958) concerning barbiturate dependence in man indicate that the criteria with respect to dosage correspond to those outlined above for the narcotic analgesics.

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With respect to species, the dog (Seevers and Tatum, 1931), the cat (Essig and Flanary, 1959) , and the mouse (Swinyard et al., 1957) readily develop dependence to sedatives. Most of the hypotheses concerning the mechanism of the development of physical dependence have not been supported by experimental evidence and will not be listed here. Recent attempts have been made t o determine whether disturbances in catecholamine metabolism in the central nervous system are related to the development of physical dependence to morphine (Gunne, 1959; Maynert and Klingman, 1962; Sloan e t al., 1963). Consistent results have not always been obtained between different species by the various investigators. The studies so far have dealt only with the catecholamine concentrations in the brain and not with rates of production and utilization, so a t present there is no evidence to support the view that disturbance in catecholamine metabolism is causally related to morphine physical dependence. Martin and Eades (1961), in a recent extension of earlier studies by Wikler and Carter (1953), reported the development of what they termed “acute physical dependence.” This phenomenon was elicited by the administration of nalorphine to dogs after an intravenous infusion of a large dose of morphine over the course of several hours. Tachycardia, hyperpnea, increased blood pressure, etc., were among the signs observed. These signs are among those observed in the morphine abstinence syndrome and the authors concluded that they represent an abstinence syndrome of mild intensity. Seevers and Deneau (1962) made somewhat similar observations in the monkey but placed a different interpretation on their findings. Noting that the sighs of stimulation resulted just as readily when nalorphine was administered prior to a large single dose of a narcotic, thus preventing the characteristic depression of morphine, as they did when nalorphine was administered after the development of depression, Seevers and Deneau interpreted their observations as evidence that nalorphine antagonizes the depressant action of the narcotics but not the stimulant actions. The signs of stimulation which resulted from the interaction of large single doses of the narcotic and an antagonist are considered to be a manifestation of the unopposed excitatory effecta of the narcotic analgesics rather than signs of abstinence. IV. Psychogenic Dependence

All drug abuse is based on a strong and continuing desire to achieve pleasure or avoid dysphoria. With all psychotropic drugs it is the primary and with some drugs like cocaine it is the only drive that leads to addiction. Although the clinical literature dealing with psychogenic or psychic dependence is enormous, only recently have laboratory studies been

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initiated to determine whether this phenomenon could be demonstrated in animals. An early indication that the answer would be affirmative was the observation by Tatum and Seevers (1929) that dogs receiving chronic cocaine injections manifested behavior which indicated a positive desire for the injections. This was apparent to these investigators even though their experiments were conducted for other purposes. Spragg (1940) reopened the question in his study of chronic morphine administration to chimpanzees. He observed that once the chimpanzees became physically dependent on morphine, they exhibited behavior indicative of the desire for morphine injections if, but only if, they were experiencing the abstinence syndrome at the time. No significant advances in this area appeared until relatively recently, when Nichols et al. (1956), Beach (1957), Wikler et al. (1960), and Weeks (1961) independently began to reinvestigate it. All of these investigators took up the problem as it pertained to morphine. This is perhaps unfortunate because the continued use of morphine leads to physical dependence, and the resultant physiological need for the drug complicates the question of whether the animal’s drug intake depends upon the drug’s positive reinforcing qualities or the negative qualities resulting from its ability to relieve the distressing abstinence syndrome. All of these investigators addressed themselves to the question of whether animals (specifically, the rat) could be operantly conditioned to self-administer morphine (or its pharmacological equivalent) to escape the distress of the morphine abstinence syndrome. Each confirmed the others conclusion that indeed the rat could be conditioned to self-administer an opiate when the reward was the relief of the distress of the abstinence syndrome. I n the language of the behavioral psychologist, this is an example of “escape avoidance” or “negative reinforcement” behavior. This finding adds another example to the long list of situations in which escape avoidance behavior can be conditioned in laboratory animals. Beach (1957) was the only one of these investigators who questioned the possibility that morphine might have a positive “euphoric” effect which could be responsible for continued opiate-directed behavior on the part of rats. His conclusions were that both the drive reduction effect of morphine on the morphine abstinence syndrome and the positive reinforcement of the euphoric effect of morphine would condition subsequent opiate-directed behavior in rats, but that the former was more influential than the latter. Recent studies in the authors’ laboratory, which have been presented at meetings of the NAS-NRC Committee on Drug Addiction but which are as yet unpublished, indicate that the monkey is a suitable subject for studying psychogenic dependence. Studies are in progress (a) in which

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the monkey is offered a free choice between solutions of psychotropic agents and tap water; ( b ) which utilize a newly developed method for prolonged intravenous administration in the minimally restrained monkey whereby a similar choice is offered between drug and placebo. It has been confirmed that monkeys are a heterogeneous population with respect to voluntary self-administration of drugs by both methods. With oral administration, some will consume large amounts of drugs regularly to the point of development of strong tolerance and physical dependence; others will drink drug solutions intermittently ; and still others refuse drugs completely. Most, but not all, monkeys will spontaneously initiate and continue the chronic intravenous administration of morphine, codeine, amphetamine, and cocaine. V. Attempts to Find Nondependence Producing Analgesics

The search for an analgesic drug devoid of morphine’s undesirable properties continues unabated. Two obvious approaches to this end are recognized. One approach is to modify the morphine molecule by substitution or simplification in an attempt to dissociate its various biological properties. The other approach is to seek an entirely new chemical agent which produces analgesia alone in contrast to the mosaic of effects produced by morphine. I n 1929, the National Research Council undertook an extensive program, formulated by its Committee on Drug Addiction, to find nonhabitforming drugs which would replace morphine for each of its legitimate uses. This program developed as a cooperative venture in the chemical synthesis, pharmacological evaluation, and clinical testing of morphine derivatives. I n all, nearly 500 derivatives of morphine were synthesized and tested in the period 1929-1941 under this program, The outbreak of World War I1 resulted in the discontinuance of this program per se, although the NRC Committee on Drug Addiction and Narcotics has continued to coordinate and to sponsor research in the various aspects of analgesia and drug abuse. One of the most dramatic events in the continued modification of the morphine molecule was the discovery by McCawley e t at. (1941) that the N-ally1 derivative of morphine was a morphine antagonist. Although the antagonistic action of N-allylnormorphine (nalorphine) to all of the narcotic analgesics is of great interest, this subject has been thoroughly reviewed by Woods (1956) and will not be dwelt upon here. Hart and McCawley (1944) demonstrated that nalorphine had analgesic properties in rats, but this finding was not confirmed by others who used slightly different techniques or other laboratory species. However, Lasagna and Beecher (1954) found that nalorphine was an effective

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analgesic in man, and Isbell (1956) demonstrated that it was nonaddicting in man. Thus, a morphine derivative was found in which the analgesic property had been dissociated from the most undesirable property of all-that of producing physical dependence. Unfortunately, nalorphine produces other undesirable effects, dysphoria and hallucinations, which are so severe as to preclude its general use as an analgesic agent. These findings changed the direction of research once it was clear that the various properties could be dissociated, Furthermore, attention was diverted from opiates and synthetic narcotic analgesics which possess most of morphine’s properties to the various members of each of the chemical classes which, like nalorphine, are morphine antagonists. WITH MORPHINE-LIKE ACTION A. COMPOUNDS

1. Derivatives and Moieties oj mmphine

Although they are completely synthetic products, the morphinan derivatives may be considered to be modifications of morphine in that the morphinan nucleus is a simplification of the morphine nucleus (see Fig. 1). Of the synthetic analgesics, the morphinans are chemically the most closely related group to morphine since they differ from the latter only by the removal of the oxygen bridge between the I and the I11 rings of the morphine nucleus. Many compounds with analgesic activity have been developed in the morphinan series, but none is distinctly different in its qualitative spectrum of activity from morphine. As in the opiate series, substitution of an ally1 group on the nitrogen yielded a nalorphine-like antagonist. May and Murphy (1954) synthesized a series of compounds which represents a further simplification of the morphine molecule. I n addition to the removal of the oxygen bridge, ring number I11 has been opened (see Fig. 1). One of these drugs, phenazocine [2’-hydroxy-2- ( N , P-phenethyl) -5,9-dimethyl-6,7-benzomorphan] is commercially available. D e Kornfeld and Lasagna (1960) found it to be 3-4 times as potent as morphine as an analgesic and to produce somewhat less sedation than morphine a t equal analgesic doses. Allyl, or other appropriate substitution, of the nitrogen atom yields compounds which are antagonists of the narcotic analgesics, as in the opiate and morphinan series. Bentley and Hardy (1963) recently prepared a new series of compounds in the morphine class, Whereas most previous modifications of morphine and synthetic analgesics resulted in more flexible molecular structures, Bentley and Hardy felt that more might be gained by making the structure more rigid. Their rationale was that the receptor for analgesia, although closely related to the receptors associated with the various

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side effech, might be sdliciently different that a very rigid molecule would have affinity only for the receptor associated with analgesia. Starting with thebaine, these investigators prepared secondary and tertiary alcohols of the tetrahydro-6,14-ethenothebaineseries as well as the corresponding 3-OH- (oripavine) tetrahydro-6,14-etheno derivatives. Some of these compounds have analgesic potencies ranging up to 10,OOO times that of morphine. Unfortunately, Deneau and Seevers (1963) have found that they possess similar potencies in suppressing morphine abstinence signs in the monkey. Although these compounds may prove to be of no practical advantage over morphine in terms of dissociation of analgesia from undesirable side effects, their remarkable potency may lead to their use as invaluable investigative tools in problems of analgesia and physical dependence. 2. Pheny Zpiperidine Derivatives

Eisleb and Schaumann (1939) reported that meperidine (see Fig. l ) , a compound with no apparent chemical relationship to morphine, produced analgesic effects in man. Although Himmelsbach (1942) demonstrated that this drug shared morphine’s capacity to induce physical dependence, the discovery provided chemists with another molecule which could be modified in their never-ending search for the ideal analgesic agent. One of the results of such chemical modifications was the development of the hexamethyleneimine series in which the six-membered piperidine ring of the meperidine series was replaced by a seven-membered ring (see Fig. 1). Throughout this entire group of compounds there has been no significant separation of analgesic activity from the undesirable properties of the narcotic analgesics. Several of the phenylpiperidine derivatives are in clinical use-primarily because of their relatively short duration of action. The short duration of action results from the fact that they are degraded by hydrolysis and dealkylation-both of which occur more rapidly than conjugation-the major route of detoxication of the opiates. It is of interest to note that substitution of the ally1 radical on the nitrogen atom of meperidine does not yield an effective narcotic antagonist. 3. The Methadone Series

Schaumann (1952), who was responsible for the introduction of the phenylpiperidine derivatives, also introduced another group of analgesic drugs, the prototype being methadone (see Fig. l ) , which bears no apparent chemical relationship to morphine. Reduction of the ketone group of this series to an alcohol results in the formation of a second asym-

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metrical carbon atom, thereby permitting the chemist to prepare a variety of optical isomers of the many potential derivatives. I n spite of intensive efforts with this group of drugs, the property of analgesia has not been separated from the many other actions of the opiate analgesics. It is of interest to note that Pohland has found that certain N-ally1 compounds in this series are morphine-like rather than nalorphine-like (1964). 4. The Thiambutenes

Adamson and Greene (1950) introduced another group of analgesics, which a t first glance do not appear to be chemically related to morphine (see Fig. 1). Although one of these, ethylmethylthiambutene (Ohton) is used clinically in Japan, these drugs offer no significant clinical advantage over morphine. 5. The Nitrobenzimidazoles

Hunger et al. (1957) synthesized a series of nitrobenzimidazole derivatives which were shown by Gross and Turrian (1957) to possess pharmacological properties similar to morphine. One of these, 1- (diethylaminoethyl) 2- (p-ethoxybenzyl) -5-nitrobenzimidazole (see Fig. 1) , is approximately 1500 times as potent as morphine, both in regard to analgesia and the undesirable properties of morphine. 6. Miscellaneozls Compounds

I n addition to the above classes of compounds, a variety of other drugs have been produced which resemble morphine, or more frequently codeine, in their over-all spectra of pharmacological actions. I n no case has effective analgesic activity been retained without the attendant undesirable effects of the opiates. Whenever the side effects have been reduced, analgesic effectiveness has likewise been reduced. The reader who is interested in extensive comparisons of the analgesic versus physical dependence properties of various individual compounds in the above series is referred to the extensive reviews of Braenden et al. (1955), Eddy et nl. (1956), and of Mellett and Woods (1963).

B. STRUCTURE-ACTIVITY RELATIONS(SAR) Repeated mention has been made above that the narcotic analgesics represented chemical modifications of morphine or that there was no apparent chemical relationship between morphine and some of the synthetic series which are pharmacologically similar to morphine. The analyses of Braenden et al. (1955) and Eddy et al. (1956) revealed that there is a common structural basis among all narcotic analgesics-natural,

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semisynthetic, and synthetic. These authors showed that the structural requirements for analgesia and for physical dependence were: (a)a tertiary nitrogen; ( b ) a central carbon atom, none of whose valences is connected with hydrogen; (c) a phenyl group, or a group which is isosteric with phenyl, which is connected with the central carbon atom. Maximum activity is obtained when the central carbon atom is connected to the nitrogen by a two-carbon chain. The authors caution that not all compounds which fulfill these requirements are necessarily analgesics or capable of producing physical dependence, but all known analgesics meet the specifications (as of 1956). It will be noted that the above authors set forth the same requirements for analgesic activity as they did for physical dependence properties. It should be remembered that this extensive analysis was made in the hope of finding chemical factors which would separate analgesia from physical dependence and that as late as 1956 no such separation was possible. With certain minor modifications, such as the acceptance of a nitrogen, none of whose valences is satisfied by hydrogen for the central carbon atom [item(b) 1, compounds like the nitrobenzimidazole derivatives, which were not known a t that time, can be accommodated under the criteria of Eddy et al. for physical dependence.

C. THENARCOTIC ANTAGONISTS Mention has already been made of the fact that allyl substitution on the nitrogen yields narcotic antagonists in the morphinan and benzomorphan series as well as in the opiates. Mention has also been made of the fact that nalorphine is an analgesic agent in man. Since the classic SAR study of Winter et al. (1957) it has been realized that N-ally1 is not the only substitution capable of yielding a narcotic antagonist in the opiate series. Following this lead, a variety of antagonists have been developed in the morphinan and benzomorphan series. Many of these have been assayed as analgesics in man. Gates, who with Tschudi (1956) was first to synthesize morphine and to confirm its structure, added another achievement by his introduction of N-cyclopropylmethyl derivatives of normorphine and l-3-hydroxyniorphinan. As antagonists, these compounds differ from the parent N allyl derivatives by possessing relatively long durations of action, e.g., 24-36 hours compared to 2-6 hours for the parent compounds. This factor alone renders the N-cyclopropylmethyl derivatives important for the treatment of narcotic overdosage in that the effect of a single dose of the antagonist will persist as long or longer than the depressant action of most of the common narcotic analgesics. The N-cyclopropylniethyl-3-

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hydroxymorphinan derivative also shows promise as a potent and effective analgesic agent at doses below those which produce hallucinations and other undesirable side effects. Archer et al. (1962) synthesized and studied a series of antagonists in the benzomorphan series. One of these, 2-dimethylallyl-5,9-dimethyl2'-hydroxybenzomorphan, appears to be an effective analgesic of about half the potency of morphine, and preliminary tests indicate that it does not produce the dysphoria and psychotic reactions which limit the usefulness of nalorphine. When tested in morphine-dependent monkeys by Deneau and Sewers (1963), this compound appeared to have no definite nalorphine-like properties as judged from its inability to precipitate morphine abstinence signs, nor did i t appear to be morphine-like as judged from its inability to suppress morphine abstinence signs. These results have been essentially confirmed in man by Fraser and Rosenberg (1963). In view of these recent encouraging findings that (a) some of the narcotic analgesics produce analgesia but do not produce physical dependence and ( b ) some of the narcotic analgesics do not produce significant dysphoria or psychotic reactions, a t least in effective analgesic doses, there is renewed hope that effective analgesic drugs which do not possess any serious side effects may soon be available. It must be emphasized that this optimism should be tempered with caution because all too often in the past, clinical experience has negated similar hopeful expressions. If this hope is realized, however, it will represent the successful culmination of over 35 years of intensive effort by many people, based on the simple premise that proper modification of the morphine molecule would result in the separation of the desirable properties from the undesirable properties. It should be pointed out that as yet no studies have been published concerning possible tolerance development to the analgesic effect of the narcotic analgesic. If tolerance does develop rapidly, the requirement for higher doses may lead to unforeseen toxic effects which could limit the usefulness of these agents. It should also be pointed out that whereas the successful development of an analgesic agent which does not produce physical dependence may prevent the iatrogenic development of a few narcotic addicts throughout the country each year, it will by no means solve the great problem of abuse of narcotic anaIgesic drugs. One of the problems associated with the search for analgesic drugs among the narcotic antagonists is that these agents produce little or no elevation of the threshold to nociceptive stimulation which is the basis of most laboratory analgesic assays. Until better laboratory procedures

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are developed for assaying analgesic activity, research in this area can proceed only as rapidly as trials in man will permit. VI. Antitussives

Substances which are effective antitussives and which completely lack the capacity to induce physical dependence have been discovered in recent years. Winter and Flataker (1954) found that narcotine was nearly as effective as codeine against experimentally induced cough in animals and that tolerance did not develop to this effect. This effect of narcotine has been confirmed in man by Bickerman et al. (1957). It is of interest to note that a century ago Claude Bernard (1864) predicted that a clinical use would be found for narcotine. Anothcr agent, dextromethorphan (d-3-methoxy-N-methylmorphinan) has been found to be an effective antitussive agent. The optical isomer of this drug is the codeine analog of the morphinan series, and morphinelike properties in general are restricted to the lev0 isomers. Benson et al. (1952) found that dextromethorphan did not possess any analgesic, sedative, or respiratory depressant properties but that its antitussive action was approximately equal to that of codeine. Many clinical studies have confirmed this finding in regard to the antitussive activity. Isbell and Fraser (1953) have confirmed the absence of other opiate-like properties of dextromethorphan. ACKNOWLEDGMENTS The research cited from the authors’ laboratory was supported by grants awarded by the Committee on Drug Addiction, National Academy of Sciences-National Research Council, from funds contributed by a group of interested pharmaceutical manufacturers, and by USPHS grants MY2814 and MY5320.

REFERENCES Adamson, D. W., and Green, A. F. (1950). Nature 165, 122. Archer, S., Albertson, N. F., Harris, L. S., Pierson, A. K., and Bird, J. G. (1962). Science 137, 541. Axelrod, J. (1956). Science 124, 263. Beach, H. D. (1957). Can. J. Psychol. 11, 104. Benson, W. M., Stefko, P. L., and Randall, L. 0. (1952). Federation Proc. 11, 332. Bentley, K. W., and Hardy, D. G. (1963). Proc. Chem. SOC.p. 220. Bernard, C. (1864). Compt. Rend. 59, 406. Bickerman, H. A., German, E., Cohen, B. M., and Itkin, S. E. (1957). A m . J. M e d . Sci. 234, 191. Braenden, 0. J., Eddy, N. B., and Halbach, H. (1955). Bull. World Health Organ. 13, 931. Butler, T. C. (1958). Federation Proc. 17, 1158. Conney, A. H., and Burns, J. J. (1962). I n “Advances in Pharmacology” (S. Garattini and P. A. Shore, eds.), Vol. I, p. 31. Academic Press, New York.

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De Kornfeld, T. J., and Lasagna, L. (1960). Anesthesiology 21, 159. Deneau, G. A., and Seevers, M. H. (1963). Unpublished observations. Eddy, N. B. (1955). In “Origins of Resistance to Toxic Agents” (M. G. Sevag, R. D. Reid, and 0. E. Reynolds, eds.), p. 223. Academic Press, New York. Eddy, N. B., Halbach, H., and Braenden, 0. J. (1956). Bull. World Health Organ. 14, 353.

Eisleb, O., and Schaumann, 0. (1939). Deut. Med. Wochschr. 65,967. Essig, C. F., and Flanary, H. G. (1959). Exptl. Neurol. 1, 529. Fraser, H. F., and Rosenberg, D. E. (1963). Personal communication. Fraser, H. F., Wikler, A., Essig, C. F., and Isbell, H. (1958). J . Am. Med. Assoc. 166, 126. Gates, M., and Tschudi, G. (1956). J. Am. Chem. SOC.78, 1380. Gross, F., and Turrian, H. (1957). Experientia 13,401. Gunne, L. M. (1959). Nature 184, 1950. Hart, E. R., and McCawley, E. L. (1944). J . Pharmacol. Exptl. Therap. 82, 339. Himmelsbach, C. K. (1942). J . Phamacol. Exptl. Therap. 75,64. Hunger, A., Kebrle, J., Rossi, A., and Koffmann, K. (1957). Experientia 13, 400. Isbell, H. (1956). Federation Proc. 15, 442. Isbell, H., and Fraser, H. F. (1950). Pharmacol. Rev.2, 355. Isbell, H., and Fraser, H. F. (1953). J . Pharmacol. Exptl. Therap. 107, 524. Isbell, H., Fraser, H. F., Wikler, A., Bellville, R. E., and Eisenman, A. J. (1955). Quart. J . Studies Alc. 16, 1. Krueger, H., Eddy, N. B., and Sumwalt, M. (1941). “The Pharmacology of thc Opium Alkaloids,” Parts I and 11, Suppl. 165. Public Health Repts. (U.SJ. Lasagna, L., and Beecher, H. K. (1954). J . Pharmacol. Exptl. Therap. 112, 356. McCawley, E. L., Hart, E. R., and Marsh, D. F. (1941). J . Am. Chem. SOC.63, 314. Martin, W. R., and Eades, C. G. (1961). J . Pharmacol. Exptl. Therap. 133, 262. May, E. L., and Murphy, J. G. (1954). J . Org. Chem. 19,618. Maynert, E. W., and Klingman, G. I. (1962). J . Pharmacol. Exptl. Therap. 135, 285. Mellett, L. B., and Woods, L. A. (1963). Progr. Drug Res. 5, 157. Nichols, J. R., Headlee, C. P., and Coppock, H. W. (1956). J . Am. Pharm. Assoc. Pract. Pharm. Ed. 45, 788. Pohland, A. (1964). Personal communication. Remmer, H. (1962). In “Ciba Foundation Symposium on Enzymes and Drug Action” (J. L. Mongar and A. V. S. de Reuck, eds.), p. 216. Churchill, London. Reynolds, A. K., and Randall, L. 0. (1957). “Morphine and Allied Drugs.” Univ. Toronto Press, Toronto, Canada. Schaumann, 0. (1952). Arch. Exptl. Pathol. Pharmakol. 216,48. Seevers, M. H., and Deneau, G. A. (1962). Arch. Intern. Pha~mncotlyn.140, 514. Seevers, M. H., and Deneau, G. A. (1963). In “Physiological Pharmacology” (W. S. Root and F. G. Hofmann, eds.), Vol. I, p. 565. Academic Press, New York. Seevers, M. H., and Deneau, G. A. (1964). In “Handbook of Physiology” (B. D. Dill, E. F. Adolph, and C. S. Wilbur, eds.), Section IV, p. 809. Am. Physiol. Soc., Washington, D. C. Seevers, M. H., and Tatum, A. L. (1931). J . Pharmacol. Exptl. Therap. 42, 217. Seevers, M. H., and Woods, L. A. (1953). Am. J . Med. 14, 546. Sloan, J. W., Brooks, J. W., Eisenman, A. J., and Martin, W. R. (1963). Psychophaimacologiu 4, 261. Spragg, S. D. S. (1940). Comp. Psychol. Monogr. 15, 132. Swinyard, E. A,, Chin, L., and Fingl, E. (1957). Science 125, 739.

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Takemori, A. E. (1961). Science 133, 1018. Tatum, A. L., and Seevers, M. H. (1929). J . Phamacol. Exptl. Therap. 36, 401. Way, E. L., and Adler, T. K. (1960). Pharmacol. Rev. 1%383. Weeks, T. R. (1961). Federation Proc. 20,397. Wikler, A., and Carter, R. L. (1953). J . Phamacol. Exptl. Therap. 109, 102. Wikler, A., Green, P. C., Smith, H. D., and Pescor, F. T. (1960). Federation Proc. IS, 22. Winter, C. A., and Flataker, L. (1954). J . Pharmacol. Exptl. Therap. 11599. Winter, C. A., Orahovats, P. D., and Lehman, E. G. (1967). Arch. Intern. Pharmacodyn. 110, 186. Woods, L. A. (1956). Pharmacol. Rev. 8, 175.

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Drugs Used in Control of Reproduction G. PINCUSAND G . BIALY Worcester Foundation for Experimental Biology, Shrewsbuv, Massachusetts

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I. Introduction

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11. Synopsis of Reproductive Physiology

A. Male B. Female. . . . . . . . . . . . . . 111. Male Fertility Control . . . . . . . . . . . A. Control of Spermatogenesis . . . . . . . . . B. Inhibition of Released Spermatozoa . . . . . . . IV. Drugs Used in Control of Female Reproduction . . . . . A. Suppression of Ovulation . . . . . . . . . B. Inhibition of Corpus Luteum Function . . . . . . C. Inhibition of Implantation and Destruction of Blaatocysts V. Future Problems and Pmibilities in the Uee of Drugs for Control of Reproduction . . . . . . . . . . . . . Addendum . . . . . . . . . . . . . References . . . . . . . . . . . . .

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I. Introduction

Recent years have brought increased emphasis to the problem of population growth. The problem has been especially acute in some of the newly emerging nations and some of the older underdeveloped ones. Although there are profound differences of opinion concerning the methods to be employed in limiting population increases, there is agreement that the welfare of individual families and that of nations would be improved if the reproductive potential were limited. The problem as such is very old. From the beginnings of written history we are reminded of various peoples’ attempts to control reproduction. The interested reader is directed to Heimes’ (1963) monograph, “Medical History of Contraception,” for a detailed account of methods used in the distant and not so distant past. Recent reviews of Jackson (1959), Jochle (1962), and Fridhandler and Pincus (1964) are also very useful sources of information concerning the topic of fertility control. The title of this presentation implies to the present authors that the discussion is to be limited to factors which limit the reproductive potential, and thus drugs which can correct an impaired reproductive process will be excluded. I n reviewing the literature on drugs that interfere with the reproductive process, one is struck by the large number of articles that have been published in this field. To accord comprehensive 286

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treatment to all the phases of the reproductive process that are affected by drugs is not within ,the scope of this presentation. The authors thus take the liberty to pass over some aspects rather lightly, and to reserve more complete treatment to those areas which seem to be most productive a t the present time. II. Reproductive Physiology

A. MALE The testes perform the dual function of elaborating the male sex hormones and producing spermatozoa. For proper function, the testes require the secretion of gonadotropins by the anterior pituitary. Spermatogenesis is dependent primarily on the follicle-stimulating hormone (FSH) , and Leydig cell function on the interstitial cell-stimulating (luteinizing) hormone (ICSH, LH). It should be pointed out that the dependence of spermatogenesis on FSH is not complete. I n hypophysectomized males spermatogenesis may be maintained by the injection of testosterone (Walsh et al., 1934; Nelson, 1941 ; Boccabella, 1963). The process of spermatogenesis in animals and in man has been reviewed extensively by Roosen-Runge (1962) and by Heller and Clermont (1964). I n most of the species the process is quite orderly and involves the definite sequence of cellular changes which have been termed the spermatogenic cycle. I n man its duration has been estimated to be about 64 days. Thus i t is evident that a drug which interferes with the spermatogenic process a t any of the various stages of differentiation ordinarily will not cause immediate suppression of sperm production. Mature spermatozoa are released into the lumen of the seminiferous tubule and then are transported by a system of ducts into the epididymis. Spermatozoa surgically removed from the epididymis are mature, as evidenced by their fertilizing ability. The biochemistry of the epididymis and epididymal spermatozoa has received increased attention in recent years. The biochemistry of spermatozoa and of accessory sexual fluids has been ably reviewed by Mann (1954) and more recently by Bishop (1961a). The gonadal-pituitary axis has been the subject of numerous investigations. Androgen-sensitive centers in the hypothalamus have been investigated (Davidson and Sawyer, 1961). Implantation of an androgen in specific hypothalamic regions results in testicular atrophy. Similar atrophy results from implants of estrogens (Michael, 1962). Direct inhibition of the pituitary by steroids is thus secondary to the primary effect on the hypothalamus, The classic gonadal-pituitary axis has thus been modified to include the hypothalamus as an intermediate.

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Spermatozoa in most mammals are ejaculated into the upper portion of the vagina and from there they are transported, via the cervix, through the uterus into the oviducts where fertilization takes place. I n most species the fertile life of spermatozoa in the female genital tract is limited to approximately 2 4 4 8 hours, The transport of spermatozoa from the vagina to the oviducts involves participation of the female genital structures, since dead spermatozoa appear in the tubes a t approximately the same time as the viable ones. Although millions or billions of spermatozoa are released a t the time of ejaculation, only a relatively small number find their way into the oviducts (Bishop, 1961a). This may explain the lowered fertility in cases of reduced sperm outputs.

B. FEMALE The growth of follicles and the hormonal interrelationships have been adequately described in numerous textbooks and research publications. For details one may consult books such as “Sex and Internal Secretions’’ (Young, 1961), “The Ovary” (Zuckerman, 1962)’ or “Marshall’s Physiology of Reproduction” (Parkes, 1950). We shall try to summarize the more recent information concerning the hypothalamic-pituitary interrelationships. The classic theory of Moore and Price advanced a direct negative feedback system between the gonads and the anterior pituitary (AP). Subsequent studies have shifted the emphasis from the direct feedback to that of an indirect one mediated via the hypothalamus. Harris (1961) has outlined the early evidence for the hypothalamic control of the AP. The primary factors were the absense of secretomotornerve fibers in the AP and the presence of the hypophysial-portal system. Subsequent work with various hypothalamic lesions and with direct hypothalamic implants of sex steroids (Bogdanove, 1963; Kanematsu and Sawyer, 1963) has pointed to the hypothalamus as the primary target organ for the ovarian feedback mechanism. More detailed information may be obtained from the excellent series of articles printed in the “Control of Ovulation” (Villee, 1961), Brain-Gonad Relationships (1962), and the recent review by Reichlin (1963). The hypothalamic areas controlling gonadotropin secretion have been localized in a number of species, and more recently the emphasis has been shifted to the isolation of hypothalamic chemical agents responsible for the secretion of the gonadotropins. The studies of Guillemin (1964), McCann and Ramirez (1964), and Nikitovitch-Winer (1962) indicate the presence in the hypothalamus of a factor or factors facilitating ovulation. The sequence of events leading to ovulation involves the production of estrogen by the growing follicle, under FSH influence; the estrogen, via the hypothalamus, stimulates the release of L H which is responsible

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for both the preovulatory swelling of the follicle and its subsequent rupture. Progesterone, produced by the corpus luteum, in turn influences the hypothalamus to shut off the secretion of LH. The control of ovulation by synthetic progestins is thought to involve the influence of the drug on the hypothalamus. This area will be more fully explored later. Following ovulation, the ovum, surrounded by some of the follicular cells, enters the Fallopian tube and is transported through the oviduct into the uterus. The process takes about 3-5 days and can be speeded up or considerably delayed by hormones or other drugs. Implantation is dependent on proper coordination of endometrial development with that of the blastocyst. Early or late arrival of the blastocyst into the uterus may result in implantation failure. The early development of the embryo is extremely sensitive to various drugs, and the maintenance of proper physiological and endocrine homestasis is of utmost importance. This brief summary of reproductive physiology deals primarily with vulnerable points that have been successfully attacked by drug treatment. 111. Male Fertility Control

A. CONTROL OF SPERMATOGENESIS 1. Hypothalamic Pituitary Suppression

With the advent of the new synthetic steroids, the possibility of their usage for controlling male fertility was attempted. Heller et al. (1958, 1959) and Apostolakis (1961) administered various progestins to men and observed suppression of spermatogenesis. At the dosages used there was a profound loss of libido, which would severely limit their usefulness in human practice. Several points which merit further discussion emerge from their study. Measurement of total urinary gonadotropins revealed certain dissimilarity in their mode of action. Steroids such as Enovid and Norlutin, which possess an estrogenic component, inhibited both spermatogenesis and gonadotropin output. However, progesterone, while inhibiting spermatogenesis, had no effect on gonadotropin excretion. It is possible that in the case of progesterone there is a certain direct effect on the testis which may supplement an effect on the pituitarygonadotropin ratio (Nelson and Patanelli, 1960). At the present time it does not appear likely that a steroid will be found with a specific antiFSH effect. I n the presence of normal ICSH (LH) levels, it is most probable that the secretion of endogenous testosterone would be of sufficient magnitude to counteract the absence of FSH. Recently, several reports have been published on a nonsteroidal pituitary inhibitor (Paget et al., 1961). This compound, la-methylallyl

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thiocarbamoyl-2-methylthiocarbamoylhydrazine (ICI-33,828), has been found to be a reversible inhibitor of pituitary gonadotropic function. From animal experiments it appears that the LH activity is inhibited more than FSH activity. The compound was not effective in all of the species studied and exhibited some toxicity. There are no reports on its use in the human male.

2. Go,nadotropin Inactivation Nonspecific antibodies against pituitary gonadotropins have been demonstrated some time ago. Likewise, the development of antihormones to gonadotropins following treatment for gonadal insufEciencies has been demonstrated. Recently results have been published concerning the effect of more specific anti-ICSH preparations in the r a t (Hayashida, 1963; Lostroh et al., 1963). Activity of this serum could be demonstrated against both exogenous and endogenous ICSH. Since both the endocrine and the spermatogenic functions of the testes were suppressed, there appears to be little doubt that libido was likewise suppressed. Suppression of libido would limit the use of immunological antihormone preparations in the human male. 3. Direct Eflsct of Drugs on the Testes

a. Temporary inhibition of spermatogenesis. On an experimental basis there exist a large number of compounds which show fairly specific temporary spermatogenic arrest. Nitrofuranes and related compounds have been studied in several species. Nelson and Steinberger (1953) have reported on the effects of Furacin (5-nitro-2-furaldehyde semicarbazone) and Furadantin [n- (5-nitro-2-furfurylindane) -1-aminohydantoin] on spermatogenesis in rats. Administration of either compound caused temporary sterility. The site of action appears to be the primary spermatocyte. It should be pointed out that the action of these compounds was relatively slow and required several weeks for the development of infertility. Furadantin is used for treatment of urinary infections in men, but Nelson and Bunge (1957) failed to observe any testicular effects in men with the therapeutic doses employed. The use of alkylating agents in male fertility control has been reviewed by Jackson (1959), and a large body of literature has appeared on the effectiveness of the various agents in this series. Needless to say, the chemical nature of the alkylating radiomimetic agents precludes their use in the human male. Species variation has likewise been quite striking. In the rat, methyl methanesulfonate and methyl ethanesulfonate (Jackson et al., 1961) resulted in 100% infertility. The site

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of action a t the spermatid and the spermatozoon was also a very desirable feature. However, in the male rabbit (Fox et al., 1963) these compounds were not effective. Further synthesis and testing in this series may yield a useful drug. During the past three years reports have appeared on a new series of antispermatogenic compounds, The compounds to be discussed include the substituted 2,4-dinitropyrroles and the halogenated diamines. Patanelli and Nelson (1964) have discussed the literature and their own results with respect to the most active dinitropyrrole, 1-(N,N-diethylcarbamylmethyl)-2,Cdinitropyrrole (ORF-1616). In rats this compound exhibited no toxicity with the doses used, but produced spermatogenic arrest a t the pachytene and resting spermatocyte levels. There was also evidence that the A-type spermatogonia were impaired. A single oral treatment of 500 mg/kg resulted in complete infertility by the twenty-first day post-treatment, and resumption of fertility by the fortyninth day. The infertile state was maintained for as long as 6 months by periodic administration of the drug with a progressive recovery subsequent to termination of treatment. Presence of normal Leydig cells and of normal seminal vesicles and prostates indicated that pituitary gonadotropin function was not suppressed. I n fact, the action of the compound on the testes was dependent on normal pituitary function. Concomitant administration of estradiol, for the purpose of anterior pituitary inhibition, and of ORF-1616 resulted in a testicular picture like that produced by estradiol alone. Selective toxicity in some animals (dog) precluded the utilization of this compound in human studies. Another series of compounds have been synthesized by researchers a t the Sterling-Winthrop Research Institute. Effects of three halogenated diamines have been investigated in various animals and men (Beyler et al., 1961; Coulston et al., 1960; Heller e t al., 1961). The Compounds Win 13099 [ N,N’-bis (dichloroacetyl) -N,N’-diethyl-l,4-~ylylenediamine], Win 18446 [N,N’-bis (dichloroacetyl) -1,8-octanediamine] and Win 17416 [N,N’-bis (dichloroacetyl) -N,iV-diethyl-1,6-hexanediamine] have been subjected to comprehensive studies in rats, dogs, monkeys, and men. The potencies and the effects of the drugs varied among different species. For example, the antispermatogenic effect developed most readily in rats; for the dog the minimal effective dose of Win 13099 was 150 mg/kg, whereas in the monkey it was 250 mg/kg. The effect of these three drugs wa8 observed only after a relatively long period of medication. I n rats, the first marked effects were observed only after 14 days of medication and comprised primarily pronounced reduction in the number of tubular spermatozoa and spermatids (Beyler et al., 1961). At 28 days of medication, the intermediate germinal elements were almost completely depleted and the tubules were lined only by a

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ring of Sertoli cells and spermatogonia. The Leydig cells did not show any apparent changes. After the treatment was discontinued complete recovery ensued in 5 to 6 weeks. I n the rat, Win 18446 was about 8 times as effective as Win 13099. I n monkeys, testicular changes were similar to the ones observed in rats. The onset of spermatogenic arrest and recovery were, however, delayed. Toxicological studies have revealed no damage to tissues other than the testes. The Leydig element appeared to be somewhat more concentrated than in normal animals. Whether this was due to simple mechanical compression following diminution of the testis, or whether there was an increase in cell number is not clear. Utilization of the Win compounds in human volunteers (Heller et al., 1961) was prompted by the extreme selectivity of the effect on the testes and by the absence of undesirable side effects. The animal studies suggested that the degree of spermatogenic damage was dose related, and that the level of spermatogenic arrest could possibly be controlled within certain limits. However, it should be repeated again that prolonged treatments of animals with all of dosages reported produced disturbances a t the primary spermatocyte and spermatogonial levels. Win 13099 was given orally twice daily a t the dosage level of 1 to 2 gm per day. The reduction in sperm counts occurred as early as the third week of medication, but in the majority of subjects it did not take place until the eighth or tenth week. With Win 18446 the dosage could be reduced to 500 mg/day, and the effect of the drug on sperm count was pronounced a t 8 to 11 weeks. These subjects had initial sperm counts of about 125 million/cm3 which were decreased to 0-4 million/cms. Not only were the counts low, but the motility was only &lo%, and there was a large proportion of abnormal cells. Unlike the experience with Win 13099, some of the subjects maintained on Win 18446 complained of slight gastric disturbances and there were alterations in hematocrit level and hemoglobin. The blood picture is being subjected to further study. The mode of action of the Win compounds is not known. Besides their action under discussion, two of them are antiamebal agents, and all three have Antabuse-like action. It is possible that the compounds interfere with some essential metabolic step during spermatogenic differentiation. The histological changes in the subjects on Win compounds have been recently described (Heller et al., 1963). I n animal studies, the compounds produced changes a t all spermatogenic levels. In humans, there was only a slight alteration from normal. Biopsies revealed primarily a decrease in the total number of spermatids and morphological changes in the maturing spermatids. All of the other testicular elements were normal. The spermatid abnormalities observed involved primarily

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nuclear and acrosomal changes. Polynucleation was also frequently seen. MacLeod (1961) has observed similar changes in semen specimens from the same subjects. Following termination of treatment, the histological picture was one of increased numbers of spermatids and decrease in abnormal forms. The semen picture correlated well with biopsies, exhibiting increased sperm count, improved motility, and decrease in abnormal forms. The hormonal picture during the diamine treatment was normal with the exception of increased urinary gonadotropins (Heller et al., 1963). I n the light of normal Leydig cell functions it is difficult to explain the increased urinary gonadotropin output. Moore et d.(1962) explained this phenomenon on the basis of the gonadotropin nonutilization hypothesis, suggesting that defective spermatid maturation resulted in lower utilization of gonadotropin. This explanation is still open to discussion. I n agreement with the results obtained with ORF-1616 in animals, the effect of Win compounds depended on the presence of adequate gonadotropin titer. Suppression of gonadotropins by stilbestrol resulted in a lowered effect of the diamines. I n concluding the discussion on the diamines, one is struck by the selectivity of their action. Due to their Antabuse-like action in subjects ingesting alcohol, their utilization in Western society is likely to be limited. I n cultures where utilization of alcohol is prohibited, the drugs might be used with safety. Another drawback could be the necessity for a low but daily regimen of the drugs. It is hoped that with chemical variants a loss of the Antabuse-like effect may be achieved. Although immunological phenomena are not generally included in discussion of drug effects, recent advances in immunologically induced infertility warrant their inclusion here. The subject has been reviewed by Tyler (1961), and at a conference on immuno-reproduction (Tyler and Laurence, 1962). With respect to male infertility produced by immunization techniques the work has been conducted primarily in animals. The injection of testicular material or of spermatozoa mixed with Freund’s complete adjuvant results, in about 2 months, in complete suppression of spermatogenesis (Katsh, 1962). The tubules are lined only by Sertoli cells and spzrmatogonia. The Leydig cells are not affected. The nature of the antigenic material is not fully understood, but i t could involve acrosomal material, hyaluronidase, and other materials. Although the reaction is achieved more easily in the presence of Freund’s adjuvant i t has been observed following repeated injections of the antigen itself (Bishop, 1961b). Davidson (1902) has reported on the induction of aspermatogenesis in men by the injection of autologous and homologous testis in Freund’s adjuvant. Biopsies on these patients a t 45 days showed sloughing of germinal epithelium; a t 5 months the lesions were more

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advanced. There are no data on the recovery phase. In guinea pigs complete recovery of spermatogenesis occurs in 6 to 8 months. E. T. Tyler (1963) has recently voiced the opinion that immunization appears to be the most likely method to succeed for nonmechanical induction of male infertility. Before the use of immunological procedures becomes feasible in human practice, additional work will have to be conducted on the safety of the adjuvant. The adjuvant is known to induce abscesses and ulcerations, and in its present form it is not acceptable for humans. At the present time, serum-induced aspermatogenesis has not been demonstrated. With respect to serum, there is the interesting observation of Rumke (1959) that sera of some sterile men contain sperm agglutinins. These serum agglutinins might have resulted from the process of autosensitization. One important aspect that emerges from the review of drugs causing temporary infertility in the male is the relatively long period of time required for achievement of infertility and for the subsequent resumption of normal fertility. In understanding the duration of the spermatogenic cycle one appreciates the reasons for this situation. At the present time no chemical or biological agents have been discovered which selectively affect the mature spermatozoa that are free within the tubule or within the epididymis. b. Permanent inhibitian. Permanent inhibition of spermatogenesis does not appear to be an attractive method for the control of reproduction. Certainly, under most circumstances, sterility is not a method of choice. The only reason for discussing this topic in this presentation is to bring to the attention the remarkable affinity of the germinal epithelium to cadmium intoxication. The problem of the acute effects of Cd on the testis was highlighted by the paper of Parikek (1960). Rats injected with 0.03 mmoles/kg of CdClz subcutaneously, show testicular changes as early as 1 hour after injection. The circulatory system of the testes is acutely affected (Gunn et al., 1963). It is not known whether the effect on testicular circulation is the primary focus of Cd action or whether it is due to primary tubular necrosis followed by release of substances from the destroyed cells which in turn account for the edema. Within the seminiferous tubule, the peripheral elements are first to be affected, but within 48 hours there is complete destruction of most cellular elements. Complete testicular necrosis may be observed within 7 to 10 days. This necrosis involves all testicular elements including the Leydig cells. Decreased androgen production causes the decrease in the size of accessory sexual organs. It is of interest that, although the germinal epithelium never recovers, the Leydig component shows signs of recovery a t about 3 months postinjection. Macroscopically, the rodent testes first becomes

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red, then bluish, and in a few days the testes become yellowed and shrunken. The dosages of CdC12 that induce the severe testicular reaction do not cause any severe pathology in other organs. I n rats one may frequently observe subcutaneous swelling a t the site of injection, but this disappears with time. For the human male the only available data are those reported by PariBek (1960) and involve accidental exposure to cadmium fumes. Pathological studies in these men a t autopsy have revealed varying degrees of testicular damage. Thus there is incidental evidence that the human testicle is not immune to cadmium damage. Mature spermatozoa are likewise very susceptible to Cd ions. White (1955) in his studies on the effect of various metal ions on mammalian spermatozoa has observed extreme toxicity of Cd to sperm. Although there appears to be little practical value to the use of CdCl, as an agent in human fertility control, it has served as a valuable tool for the investigation of various biochemical interrelationships, Gunn and his co-workers (1955, 1961; Gunn and Gould, 1956) have published extensively on the interrelationship between cadmium and zinc, and have used the testicular damage produced by cadmium for determination of functional states. The competition between Cd and Zn (Cotzias et a.!., 1961) has been used to clarify the dependence of normal testis function upon zinc. I n the female rat there is no damage rendered to the ovarian tissues by CdC1,; it has been observed that the female rat and mouse tolerated cadmium much better than males. I n speculating on the effect of Cd on the testes and mature spermatozoa, there exists the remote possibility of finding combinations of cadmium with other ions or other biological materials which might induce only transitory damage to the germinal epithelium. Since White (1955) has shown the extreme toxicity of Cd to ejaculated spermatozoa, it would be of extreme interest to study the effect of very low doses of Cd, insufficient to cause testicular damage, on the metabolism, motility, and fertility of epididymal or ejaculated spermatozoa. I n this respect Kar et al. (1961) have noted no effect of Cd on fertility in the rat, when the dosage used caused only slight tubular necrosis. However, the epididymal concentration of Cd following a single small dose must be lower than the effective concentrations used by White (1955) in his in vitro studies.

B. INHIBITION OF RELEASEDSPERMATOZOA 1. Vaginal Spermicidal Agents

The field of intravaginal contraceptive agents is extremely broad and has been reviewed extensively in the past several years by Hartman

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(1959), Tietze (1960b, 1963), and MacLeod et al. (1961). Several factors must be considered in preparation of these agents: (1) physical properties of the finished product, (2) the spermicidal characteristics, and (3) subject acceptance of the product. Needless to say a product that is not acceptable to a large segment of patients, for whatever reason, will not be an effective agent in the control of reproduction. From the standpoint of physical properties, the product must be (a) of such composition as to impede effectively the progress of spermatozoa, ( b ) evenly distributed throughout the vagina, and ( c ) of nonirritating nature to either the male or the female. The chemical nature of spermicidal agents has been reviewed in extenso (Mann, 1958). Basically, the agents used serve as inactivators of sperm motility and thus supposedly render the spermatozoon incapable of causing fertilization. The preparations are tested in vitro for their sperm-inactivating potency by several methods, with the hope that the in vitro assessment will compare favorably with the in vivo performance. It is of interest that a product tested by several in vitro techniques (MacLeod et al., 1961) has scored well in a more physiological study (Johnson and Masters, 1963). The effectiveness of the intravaginal agents is limited in practice. The failure rates as reported by Tietze (1960a) range from 11 to 40%, which a t the present time would not qualify the agents as good means for controlling reproduction. Chang (1960) has tested several intravaginal preparations in rabbits and found them to be of little use in inhibiting fertility. 2. Miscellaneous Inhibitors

I n considering drugs used for the control of male reproduction, it would be extremely advantageous to find some that might render epididymal spermatozoa infertile. Such drugs would be safer if there were no damage to the germinal epithelium or any possibility of inducing undesirable mutations during the spermatogenic cell division. At the present time, drugs of this nature have not been discovered. The only reference to drugs affecting epididymal sperm directly (Jackson, 1959, p. 146) is to certain ethyleneimino compounds ; however, these agents lack specificity and are known to exert damage on the germinal epithelium. Following the demonstration of hyaluronidase in mammalian spermatozoa and the speculation of its possible action, a number of studies were undertaken on the role of the enzyme in fertility. I n well-designed experiments no correlation has been found between enzyme inhibition and antifertility properties (Jackson, 1959). Another agent that was claimed to effect animal and human fertility is m-xylohydroquinone (Senyal, 1962). However, recent studies (Kar et aZ., 1963) have not confirmed the original claims.

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IV. Drugs Used in Control of Female Reproduction

The production of ova is a cyclical phenomenon, the various stages of which may be distinguished by diverse methods. The literature dealing with this subject is extremely abundant, and no attempt will be made to include all of the published reports. This discussion of the control of female reproduction will be limited to physiological phenomena that precede placentation ; abortifacient drugs will not be discussed.

A. SUPPRESSION OF OWLATION 1. Steroidal Ovulation Inhibitors One of the more revolutionary breakthroughs in the field of human fertility control has been the discovery of very potent steroidal ovulation inhibitors. The original observation of Makepeace et al. (1937) that progesterone inhibited ovulation in the rabbit was substantiated by Pincus and Chang (1953). I n women, 300 mg of progesterone per day taken orally resulted in ovulation inhibition in 80% of cases (Pincus, 1956). The high dosage and frequent incidence of breakthrough bleeding limited the practical application of the method. Subsequently, the utilization of potent 19-norsteroids, which could be given orally, opened the field to practical oral contraception. All preparations used for contraception contain a synthetic progestin as the major component (Fig. 1) and an estrogen as the minor (by weight) component (Fig. 2). The original cyclic use (Pincus, 1955) from day 5 to 25 of the menstrual cycle is the regimen employed. A t the present time the study of oral contraceptives is a full-time job. I n this review we shall refrain from a systematic literature coverage of all preparations and stress rather the studies dealing with the mode of action, effects on hormonal homeostasis, and on side effects. From the studies on ovulation inhibition in the rabbit, Pincus and co-workers have concluded that lack of ovulation is caused by the suppression of pituitary LH release mechanism. The studies of Sawyer and Kawakami (1961) have clearly shown that ovulation suppressants produced, in the rabbit brain, an increase in the EEG after-reaction threshold, which they interpreted as being indicative of ovulation inhibition. There was a differential effect on the arousal threshold-estrous behavior. Progesterone and Delalutin tended, over-all, to elevate the arousal threshold, while norethynodrel, Norlutin, and Nilevar did not change the EEG arousal threshold. The importance of this finding is seen in the numerous publications that state that there is no apparent affect of ovulation suppressants on libido. Similar findings have been published by Kobayashi

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Norethindrone

Norethynodrel

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Norethindrone acetate

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Lynestrenol

Ethynodiol diacetate

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PIQ.1. Progestins used in oral contraception.

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Ethynyl estradiol

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Ethynyl estradiol, 3-Methyl ether (Mestranol)

FIQ.2. Estrogens used in oral cont,raception.

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et al. (1962) for the rat. In comparing several areas of the brain following administration of progesterone, the area most affected, in terms of activation levels, was the posterior hypothalamus-archicortical-activating system. The posterior hypothalamus is believed to be the area responsible for ovulation control. While the most probable mode of action of steroidal ovulation suppressants is the inhibition of LH release, we must examine the available evidence for this concept. In parabiotic rats (Epstein et aZ., 1958) there is evidence that ovulation-suppressing steroids reduce the gonadotropin output. I n the rabbit there is excellent evidence that ovulation suppression is the result of failure of LH release rather than being due to the failure of the follicle to respond. Administration of gonadotropins to rabbits treated with ovulation inhibitors results in normal ovulation (Edgren and Carter, 1962; Harper, 1962). I n normal rats, prolonged treatment with norethynodrel did not completely inhibit ovulation, as evidenced by the presence of corpora lutea (Holmes and Mandl, 1962a), although fertility was completely suppressed. One thus must be careful in differentiating the terms ovulation, inhibition, and contraception. In the mouse (Purshottam et al., 1961) and the rat (France and Pincus, 1964) certain ovulation-inhibiting steroids reduce the number of ova superovulated by gonadotropin. To incorporate this finding into the physiological assessment of the mode of action of ovulation-inhibiting steroids is extremely difficult, because the drugs were used a t levels higher than those required for production of infertility, and the number of ova shed was above normal. Nevertheless there is indication that the estrogens in particular may have an effect a t the level of the ovary or the follicle. This has been shown by the reduction of superovulation in gonadotropin-injected hypophysectomized rats. However, heavy doses of purified norethynodrel did not inhibit the ovarian sensitivity in immature intact rats to exogenous gonadotropin stimulation (Eckstein and Mandl, 1962),nor did they inhibit ovulation in hypophysectomized rats (France and Pincus, 1964). It appears then that the interpretation of results depends to a certain degree on the end point that is chosen. The data for the human female are somewhat contradictory. Part of the difficulty lies in the inadequacy of assays for human pituitary gonadotropins; most assays consist merely of measurement of total urinary gonadotropins without separation into the FSH and LH fractions. Nelson and Patanelli (1960)have expressed the opinion that the effect of ovulation-inhibiting compounds may depend not necessarily on total gonadotropin suppression as much as on the upset of the LH-FSH balance. Such imbalance would then be responsible for lack of follicular matura-

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tion and of ovulation. Limited data a t the present time favor the concept of selective gonadotropin inhibition. Paulsen et al. (1960) found no decrease in the excretion of total gonadotropins in the urine of menstruating women injected with progesterone. They employed immature rat ovaries as the end points and in this assay there is no differentiation between FSH and LH. Studies of Brown and co-workers (1962; Brown and Matthew, 1962) indicate that gonadotropin excretion was normal in women taking oral contraceptives. They employed the immature mouse uterus as the end point. Loraine et al. (1963) observed no decrease in urinary gonadotropins in women on extended ovulation depression therapy, and interpreted their findings as a n indication of the ovarian site of action of these compounds. I n postmenopausal women with high urinary gonadotropin excretion, there was a depression in urinary gonadotropin output following administration of various ovulation-inhibiting steroids (Brown and Matthew, 1962; Loraine, 1964). Several studies are not in agreement with those mentioned above. Demo1 and Ferin (1964) administered a different progestational agent to normal subjects and observed a significant drop in urinary gonadotropins. Walser and co-workers (1964) observed a variable result. In some patients there was a decrease and in others there was no decrease in urinary gonadotropins. Buchhoh et al. (1964) have observed a decrease in urinary gonadotropins in both postmenopausal and menstruating women taking norethisterone. I n their study there was a consistent midcycle increase in gonadotropic excretion which was associated with ovulation. This increase was abolished during the norethisterone-treated cycles. How does one account for the differences observed? Differences in assay procedures have been previously mentioned, but individual differences do contribute to the variation. I n Walser’s e t al. series (1964) there was both inhibition and no effect. Since the question of gonadotropic secretion is of primary importance in understanding the mode of action of ovulation-suppressing steroids, it appears that a cooperative effort is in order to resolve the differences. Likewise, with the different agents, there may be a differential effect on the urinary gonadotropin output. During the normal cycle, there are two peaks of estrogen excretion, one prior to ovulation and the second one during the luteal phase. Pregnanediol excretion is limited to the luteal phase and serves as an indicator of the progesterone output by the corpus luteum. In the absence of the corpus luteum (CL) the output of pregnanediol is very low. All of the above reports agree that during treated cycles in which ovulation is thought to be suppressed, there was a significant decrease in estrogen and pregnanediol excretion. It is not likely that the ovulation-

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suppressing steroids interfere with CL function once it has become established. Vanek (1964) observed only a slight decrease in pregnanediol excretion in women who took ethynodiol diacetate (2 mg/day) from day 17 to day 24 of the cycle. Pincus et al. (1958) have reported the same for Enovid users. With respect to previous animal studies, evidence was presented indicating that a peripheral action of steroidal ovulation suppressants was one of the possible modes of their action. For the human female the results are somewhat scanty. Lunenfeld and co-workers (1963; Lunenfeld, 1964) have administered gonadotropins to amenorrheic women and have observed increases in the excretion of estrogens and pregnanediol characteristic of a normal menstrual cycle. In 2 out of 3 patients who were given gonadotropins together with an ovulation inhibitor, there was no rise in the steroid excretion. The author interpreted the results as indicating that the ovulation suppressant used (601-methyl-17a-acetoxyprogesterone) was acting a t the ovarian level. Hecht-Lucari (1964), using a different steroid, observed similar results in one patient. I n the same paper the author also gives a good summary of results for and against the motion of peripheral action of ovulation inhibitors. Staemmler ( 1960), on the other hand, failed to observe blocking action of norsteroids in humans injected with gonadotropins. Ovulation inhibition in the human is generally ascertained by indirect physical or chemical methods. Direct observations of ovaries during owlation-inhibited cycles are much more limited. Garcia and Pincus (1964) observed no signs of recent corpora lutea in several treated patients. Matsumoto e t al. (1960) found no signs of ovulation in 11 patients. Their data have been subjected to criticism (Holmes and Mandl, 1962b) for a number of reasons, but they nevertheless felt that absence of corpora lutea was due to the action of the ovulation-suppressing steroid. gstergaard (1964), in a series of 33 normally menstruating women, observed no ovulations at laparatomies performed on the twenty-fourth day of the cycle, when treatment with a steroid was initiated on the fifth day of the cycle. Ovarian biopsies showed normal follicles and stroma and remnank of old corpora lutea. Lauweryns and Ferin (1964) have likewise observed no active or involutive corpora lutea in 3 patients maintained on oral contraceptives for from 7 to 9 months. Ovulation inhibition appears to be the primary action of steroidal oral contraceptives. However, Goldzieher et al. (1962) have observed that in 6 out of 80 norethindrone-treated cycles, pregnanediol excretion was at a level found during the normal luteal phase. This finding was interpreted as being indicative of ovulation and CL formation. Extrap-

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olation of these data to cover all of the subjects in their study yielded 424 potentially fertile cycles, but there were no pregnancies.

Numerous studies on the effect of oral contraceptives on the endometrium have been reported (Jackson, 1962; Pincus et al., 1958; RiceWray et al., 1963). The endometrial response has been shown to vary with respect to the various agents used and according to the number of cycles of therapy. Endometrial biopsies taken from women on norethynodrel therapy (Pincus et al., 1958) reveal a secretory response a t the early stages, with a rapid involution of the glands thereafter. There is edema of the functional layers of the endometrium and a moderately loose stroma. The generalized response could be termed as being pseudodecidual. Biopsies from subjects on norethindrone (Rice-Wray et al., 1963) show a more hypoplastic endometrium that remains fairly stable during the treatment cycle. There are only a few small glands, and the stroma tends to be dense and fibrillar. I n patients on norethindrone therapy for extended periods of time, the endometrium becomes remarkably stable after three cycles of therapy, with little change after additional cycles, The differences observed between the two most widely used agents, norethynodrel and norethindrone, most likely reflect the differences in their biological activities. Norethynodrel tends to exhibit slight estrogenicity in addition to its progestational activity, whereas norethindrone is a fairly strong antiestrogen. From the above discussion it becomes apparent that the endometria of subjects on oral contraceptives are not synchronous with ones of the normal cycle, and even if ovulation and fertilization were to take place the blastocyst probably would not encounter an endometrium favorable to implantation. Another factor that is frequently discussed with respect to ovulation inhibitors is their effect on the quality and quantity of cervical mucus. Under progestational influence the mucus becomes viscous, crystalizes upon drying, and the spinnbarkeit (the ability to form a thread) is considerably lowered. In vitro tests on sperm penetrability of such mucus show poor penetrability which would tend to obstruct passage through the cervix (Zafiartu, 1964). Postcoital tests on subjects on norethynodrel reveal motile sperm in about 50% of the cases; in subjects taking other progestational agents the mucus is more viscous and only few motile sperm are seen (Jackson, 1963). The three factors that have been discussed, (1) failure to ovulate, (2) change in endometrial histology, and (3) changes in cervical mucus, could work additively to prevent fertilization and implantation. Safety of any agent is a primary prerequisite to its use in human therapy. All the reports (Pincus, 1961, 1964; Garcia and Pincus, 1964;

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a. BIALY

Swyer, 1964) concerning laboratory studies on blood and urine reveal only minor deviations from normal. One aspect of ovulation inhibition that has received recent attention is its relation to thromboembolic phenomena. Careful statistical studies (Searle and Co., 1962) have failed to reveal any increase in the incidence of thromboembolism in women taking steroidal oral contraceptives. Blood studies have revealed a decrease in the clotting times (Pincus, 1964; Sobrero et al., 1963), but the values were still within normal ranges. Blood chemistry has also been investigated. A number of factors implicated in the blood-clotting mechanism are elevated, suggesting a condition of blood hypercoaguability (Egeberg and Owren, 1963; Mammen et d.,1963; Brehm, 1964; Pelgram, 1964). However, factors responsible for fibrinolytic activity are likewise elevated (Brehm, 1964). Brehm (1964) is of the opinion that the circulatory physiology of his subjects was normalized, and if there was any change i t was toward inhibition of thrombosis rather than the opposite. Garcia and Pincus (1964) have reported the reduction in superficial varices in women on birth control pills. It has been repeatedly emphasized that a t the present time there are no data that can link either the blood coagulation picture or varices to cases of spontaneous, injuryfree thrombophlebitis. Another aspect that has received considerable attention has been fertility following termination of oral contraception (Goldzieher, 1964 ; Pincus, 1964). The endometrium returns to normal in one or two cycles after termination of therapy, and the subsequent fertility is normal if not increased.The ovaries exhibit no permanent damage in that the number and morphology of primary follicles is not disturbed (Rock et al., 1957). I n laboratory animals (Lakshman and Nelson, 1963), there was a n indication of a rebound phenomenon following ovulation suppression, and no signs of diminished fertility, following extended periods of treatment with ovulation-suppressing steroids (Lipschutz et al., 1963). Steroid excretion following termination of therapy appears to be normal (Loraine, 1964). There is no evidence that the drug therapy increases the incidence of genital cancers. Papanicolaou smears and endometrial biopsies, in fact, show a reduction in positive cases (Garcia and Pincus, 1964; Tyler, 1964). Adrenal function during treatment with ovulation inhibitors has been the subject of several studies. Layne et al. (1962) have shown that plasma cortisol levels were increased during norethynodrel therapy. This is not surprising in view of the compound’s estrogenicity and the known tendency of estrogens to increase the cortisol-binding capacity of plasma. When Enovid was administered to the subjects there was a small but significant drop in cortisol secretion. It has been questioned whether

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the increase in the total plasma cortisol may be harmful to the subject (Metcalf and Beaven, 1963), and adrenal function following extended drug therapy has been investigated. Concomitantly with the increased plasma cortisol levels, there is a decrease in the excretion of urinary 17-ketosteroids and 17-hydroxycorticosteroids (Wallach et al., 1963). Although the decrease in excretion is noticeable it is not statistically significant. I n subjects on oral contraceptives for a number of years, the response to exogenous ACTH appears to be normal Side reactions are known to occur in subjects taking oral contraceptives. Publications dealing with steroidal oral contraceptives invariably deal with this problem. The whole topic may be summarized as follows. Every steroidal drug that has been utilized has caused side reactions in the subjects. The problem is most severe during the early medication cycles, with improvement in the following ones. I n many instances, the side reactions are severe enough to cause the subjects to withdraw from the pill-taking program. Certain side effects are d a c u l t to evaluate objectively and Pincus (1961) has stressed the importance of the psychogenic element when interviewing is being conducted. It would appear that among the subjects one finds individuals who report subjective improvement in their well-being as well as the ones who report deterioration. Differences among the populations used in the field trials are also responsible for varying degrees of total reactions (Jackson, 1963; Goldzieher, 1964), and direct comparison of different compounds utilized in different geographical areas is difficult. One of the avenues utilized in trying to reduce the incidence of side reaction has been the reduction in the daily dosage of the various drugs. The common experience has been that when the progestin dose was reduced, the dose of the estrogen has to be increased in order to assure proper cycle control and safety. Determination of the proper progestin-estrogen ratios in oral contraceptives has been somewhat arbitrary (Jackson and Linn, 1964) and more research is required in this direction. Another approach toward reduction of side effects has been the synthesis of new progestins. There are indications (Pincus et al., 1962; Andrews and Andrews, 1964; Mears, 1964a) that certain new progestins have a lower incidence of side effects than norethynodrel and norethindrone. Interestingly, whereas research on new progestins has been very active, the estrogenic components have been quite uniform in the majority of formulations. The two estrogens utilized by the various drug manufacturers are ethynyl estradiol and its 3-methyl ether (mestranol) . Metabolic transformation of both norethynodrel and norethindrone yields a number of metabolites with estrogenic properties (Brown and Blair, 1960; Langecker, 1961; Layne et al., 1963). Ethynyl estradiol

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has been identified as one of the metabolities. I n view of the known pituitary-inhibiting effects of the estrogens, it has been suggested that in the case of norethynodrel and norethindrone it is the estrogenic metabolite (ethynyl estradiol) that is the active ovulation inhibitor rather than the progestational component (Klopper, 1961). The fact that a metabolite possesses antiovulatory activity does not in itself exclude the possibility that the parent compound also has similar activity. In the rabbit, Pincus (1961) has reported that ethynyl estradiol was not an ovulation suppressant, in contrast to the marked ovulation-suppressing activity of both norethynodrel and norethindrone. Some of the newer progestins are not likely to undergo transformation to estrogens, and they do act as “apparent” ovulation inhibitors in the human (Ferin, 1962). Some years ago Goldzieher e t al. (1947) reported ovulation inhibition in the human with therapeutic doses of estrone sulfate and stilbestrol. However, after several cycles of inhibition, the pituitary exhibited an escape phenomenon and ovulation ensued. Reasoning from the preceding discussion of estrogenic metabolites of the norsteroids and from data just reported, Goldzieher and co-workers (1963) instituted a contraceptive regimen involving the intake of an estrogen (mestranol; see Fig. 2) for the first 15 days and then for the next 5 days a combination of the estrogen with a progestin (sequential therapy). The claim is made that such a treatment is more physiological than the progestin-estrogen complete cycle therapy. Unlike estrone sulfate and stilbestrol, ethynyl estradiol and its 3-methyl ether do not produce pituitary “escape” even after prolonged usage. Low urinary pregnanediol values in a number of patients indicate absense of luteal function and presumably, therefore, a pituitary inhibition. Goldzieher reports (1964) that the sequential regimen has proved effective in over 15,000 cycles. The endometrium during the estrogen administration period exhibits a well-developed proliferative phase, which changes to resemble a normal 19-day endometrium following the 5 days of estrogen plus progestin therapy. In Goldzieher’s study more women showed a decreased flow than an increased flow after termination of the treatment cycle. Mears has recently reported (1964b) that among her patients there were more with increased than with decreased menstrual flow. The differences reported by the two investigators may reflect differences in patient numbers and differences in populations. Another side effect that must be considered is the possibility of increased gastrointestinal problems with extended periods of estrogenic stimulation. If the future proves sequential therapy to be safe and effective, the cost of oral contraception may be reduced. In conclusion it may be stated that the efficacy of steroidal oral con-

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traceptives has been established beyond any doubt. The acceptability of the products has been demonstrated. There do not appear to be any striking qualitative differences among the numerous products available, and the differences in subject numbers utilizing the various products do not reflect a t the present time the physiological merits of the drugs. 2. Nonsteroidal Agents Influencing Hypothalamic Pituitarg Axis

Several reports have recently appeared on the efficacy of la-methylallylthiocarbomoyl-2-methylthiocarbomoylhydrazine (ICI 33828) in reducing pituitary gonadotropin activity in several groups of animals (Brown, 1963). ICI 33828 was administered to postmenopausal women, and a significant reduction followed in the excretion of urinary gonadotropins (Brown et al., 1963). I n premenopausal women the compound, when administered from the fifth through the twenty-fifth day of the cycle, inhibited luteal function and the excretion of pregnanediol and estrogens without a concurrent reduction in urinary gonadotropins (Bell e t al., 1962). Administration of the drug during the luteal phase (days 19 through 25), had no effect on luteal function. The excretion of urinary steroids during administration of this compound resembles the condition observed during administration of steroidal ovulation inhibitors. The presence of certain undesirable side effects tends to limit at the present time the usefulness of this compound for practical ovulation control, but this may be controlled by lowering the dose of the drug and ascertaining the lower limits of ovulation inhibition,

B. INHIBITION OF CORPUS LUTEUMFUNCTION A variety of agents have been utilized for the inhibition of CL function (Gaunt e t al., 1963). The most promising one appears to be that of Shelesnyak and co-workers. These workers have established that a variety of ergot-alkaloids prevented the development of the decidual reponse (Shelesnyak, 1956;Carlsen et al., 1961). The effect of the alkaloids could be counteracted by the administration of exogenous progesterone or luteotropin (Shelesnyak, 1956; Zeilmaker and Carlsen, 1962). From experiments on laboratory animals i t becomes apparent that the effect on CL function was indirect and was mediated by the effect of the drug on the release of gonadotropin. The effect was only transient and pituitary function was restored to normal shortly after administration. Shelesnyak’s group has undertaken to study the effect of ergocornine on the luteal phase of the human menstrual cycle (Shelesnyak et al., 1963). Five women who showed the ovulation-associated rise in basal body temperature were given 2 mg of ergocornine methanesulfonate by mouth. Urine samples were obtained prior to and after ergocornine administration

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and were analyzed for pregnanediol, estrogens, and corticoids. The postovulatory rise in both pregnanediol and estrogen was indicative of ovulation, Following ergocornine, the 24-hour urine samples had lowered pregnanediol and estrogen levels and a concurrent rise in 17-ketosteroids and 17-hydroxycorticosteroids. The lack of urine gonadotropin analysis makes interpretation somewhat difficult. The drug might have produced the observed effect either by indirect via pituitary, or direct corpus luteum action. By interfering with progesterone output the drug may prevent proper decidualization and nidation and in this manner induce infertility.

C. INHIBITION OF IMPLANTATION AND DESTRUCTION OF BLASTOCYSTS Nidation is preceded by a well-defined sequence of endometrial changes. Interference with these changes leads to failure of implantation. Several drugs are known to interfere with nidation and can be considered from both practical and experimental viewpoints. Banik and Pincus (1962) tested several antiprogestins for implantation inhibition. The compounds were administered to rats and mice, following mating for 3 days. Several of the compounds were very effective in inhibiting implantation in both rats and mice. However, there was no correlation between antiprogestational activity in the rabbit and anti-implantation activity in rats and mice. The compounds had no effect when administered on the eighth day or later. It is also of interest that even when implantation was inhibited by the early administration of the drugs, there was no deleterious effect on blastocysts. Among the antiprogestins the most conspicuous arc the estrogens. The antifertility effects of estrogens when administered after mating have been demonstrated in laboratory animals. The postulated mode of action has been either the retention of ova in the tubes or their complete expulsion from the uterus. The known effect of estrogens in increasing myometrial contractility might be responsible for expulsion of the ova. Several nonsteroidal antifertility agents which are derivatives of diphenylhydronaphthalene and of 2g-diphenylindene have been reported to have deleterious effects when administered to rats and rabbits (Duncan et at?., 1962; Duncan and Lyster, 1963; Chang, 1964). Chang has shown that triethylamine causes degeneration of tuba1 ova, and this might account for the antifertility effect of the above compounds. Their biological activity a t the antifertility level is confined primarily to the antiestrogenic effect (Duncan and Lyster, 1963). Another nonsteroidal antiestrogen, ethanoxytriphetol (MER-25), has been shown to possess antifertility effects in rats and rabbits (Segal and Nelson, 1958; Chang, 1959). The compound interferes with the normal development of fertilized

DRUGS USED IN CONTROL OF REPRODUCTION

307

ova during their passage through the Fallopian tubes (Chang, 1959). Triethylamine has been found to have no effect on blastocyst survival in utero (Chang, 1964). From this short description it appears that the relatively short period of time during which the nonsteroidal antiestrogens are effective would limit their usefulness in the human female, where the time of ovulation and fertilization is much less clearly defined. The antiovulatory effects of norethynodrel have been clearly established. However, it has been demonstrated by Davis (1963) that norethynodrel when administered to rats for 3 days postcoitus effectively blocked pregnancy. The effect is most likely on the tuba1 ova since effect of the drug was much less pronounced on uterine blastocysts. I n the rabbit it has been demonstrated that norethynodrel administered on days 1,2, and 3 after artificial insemination caused a drastic reduction in the number of normal blastocysts on day 6 (Chang, 1964). Administration of similar high doses a t the time of implantation had no effect on pregnancy. In both species the doses used considerably exceeded the antiovulatory doses and the effects may be attributable to the inherent estrogenicity of the compound. V. Future Problems and Possibilities in the Use of Drugs for Control of Reproduction

The general acceptance of the steroidal oral contraceptives raises certain problems that require further clarification. The foremost one is the problem of safety following long-term usage of the drugs. At the present time there is no positive evidence concerning health hazards to subjects employing this form of fertility control. The lingering doubts produced by the thromboembolic controversy should be followed up. In view of the large potential market for these products, further studies concerning their effect on various physiological functions must be continued. Since the initiation of the birth control programs, there has been a steady decline in the dosages of the drugs that are necessary to control fertility. This has been accomplished via (1) alteration of the progestinestrogen ratios and (2) synthesis of new more powerful ovulation inhibitors. It is most probable that the newer steroidal drugs will replace the ones presently used. The reduction in dosage is most welcome because it reduces the chances of upsetting the inherent hormonal homeostasis, as well as the cost of the product. Nonsteroidal ovulation inhibitors are being investigated with increased interest. Although a t the present time there does not appear to be a product useful for human utilization, there have been important breakthroughs in the field which may lead to synthesis of new drugs. Drugs operating a t the level of the ovary or the ovum have been

308

G . PINCUS AND G. BIALY

discovered. This would appear to be a fruitful area of research in that the female’s own periodicity need not be interferred with. The questions of effectiveness and safety will have to be investigated. Unlike the steroidal ovulation inhibitors, some of the other drugs have exhibited varying effectiveness in different animal species. Whether they are effective in the human female is not known. The necessity for daily ingestion of contraceptive pills creates certain problems to their users. Formulation of a drug that would necessitate intake a t more prolonged intervals might be advantageous, but the habit of daily pill-taking may be more useful than the need for remembering a specific time or frequency per month. Immunization procedures, in both the male and the female, for induction of temporary infertility appear to be very attractive. Isolation of potent and specific antigens from spermatozoa or testes may be anticipated, as well as preparation of more adequate adjuvants. The outlook for immunological control of fertility appears to be more promising in the male than in the female. The economic and practical aspects of immunization procedures might make them more utilizable in areas of low economic and educational background. In reviewing the question of drugs used in the control of reproduction, it becomes evident that the relative merits of any drug can be determined to a great extent by the user’s ability to follow instructions. The simpler the procedure for drug utilization, the more readily it becomes acceptable to the population. Unfortunately, in the areas where population control is most urgent, the educational levels are rather low. It thus becomes apparent that dispersion of knowledge must be the first line of offense. At the present time a number of steroidal ovulation inhibitors are being utilized in various areas of the world with gratifying success. Cost of the products has been diminishing steadily, making the products more easily procurable. With increased competition among t.he various drug companies the price may be further lowered. Each new product must be submitted to adequate testing procedures to assure maximum safety. Work must be continued to eliminate undesirable side effects which are severe enough in certain instances to limit the use of the product. Longterm effects of prolonged drug therapy should be kept in mind, and a certain degree of reservation may be a valuable safety factor. It is conceivable that with increased number of subjects new evidence on side effects may be forthcoming. It is imperative that such information be disseminated among investigators in the field of reproductive physiology so that the problem can be properly evaluated. It must not be overlooked, however, that improved menstrual function accompanies the cyclical use of the progestin-estrogen combination, and that this, along with

DRUGS USED IN CONTROL OF REPRODUCTION

309

accompanying physiological regularization, may lead to important longterm benefits. There is no question but that practical aspecb of fertility control have given an added impetus to the whole field of study of reproductive physiology. It is only through the understanding of the normal processes that future progress in various areas of control may be expected. It is hoped that a headlong search for new drugs will not obscure the continuing need for basic research. ADDENDUM Readers interested in further surveys of literature dealing with the field of reproduction and its control are directed to the following eources: (1) Journal of Reproduction and Fertility, (2) International Journal of Fertility and Sterility, (3) Bibliography of Reproduction, (4) Annual Reviews of Physiology and Pharmacology, (5) Proceedings of the International Planned Parenthood Federation, and (6) Proceedings of National and International Societies of Fertility and Sterility. From time to time there appear publications of various academies dealing with aspects of reproduction (Greep, 1963).

REFERENCES Andrews, W. C., and Andrews, M. C. (1964).Fertility Sterility 15, 75. Apoetolakis, M. (1961).Acta Endocrinol. 37, 75. Banik, U.K.,and Pincus, G. (1962).Proc. SOC.Ezptl. Biol. Med. 111, 595. Bell, E. T., Brown, J. B., Fatherby, K., and Loraine, J. A. (1962). Lancet 11, 528. Beyler, A. L., Potts, G. O., Coulston, F., and Surrey, A. R. (1961). Endocrinology 69, 819. Bishop, D. W. (1961a). In “Sex and Internal Secretions” (W. C. Young, ed.), p. 738.Williams & Wilkins, Baltimore, Maryland. Bishop, D. W. (1961b).Proc. Soc. Exptl. Biol. Med. 107, 110. Boccabella, A. V. (1963).Endocrinology 72,787. Bogdanove, E. M. (1963).Endocrinology 73,696. Brain-Gonad Relationships (1962). Intern. Congr. Physiol. Sci. 18nd, Leyden, 1962 p. 603.Excerpta Medica Foundation, New York. Brehm, H. (1964).Intern. J . Fertility 9,45. Brown, J. B., and Blair, H. A. F. (1960).Proc. Roy. SOC.Med. 53, 433. Brown, J. B., and Matthew, G. D. (1962). Recent Progr. Hormone Res. 18, 337. Brown, J. B., Fatherby, K., and Loraine, J. A. (1962).J . Endocrinol. 25,331. Brown, P. 5.(1963).J. Endocrinol. 26,425. Brown, P. S.,Tulloch, M. I., Crooks, J., Klopper, A. I., and Thorburn, A. R. (1963). Brit. Med. J . 2, 1630. Buchholz, R., Nocke, L., and Nocke, W. (1904).Intern. J . Fertility 9,231. Carlsen, R.A., Zeilmaker, G. H., and Sheleanyak, M. C. (1961). J . Reprod. Fertility 2, 369. Chang, M. C. (1959).Endocrinology SS, 339. Chang, M. C. (1960).Fertility Sterility 11, 109. Chang, M. C. (1964). Fertility Sterility 15, 97. Cotzias, G.C., Borg, D. C., and Selleck, B. (1961).Am. J . Physiol. 201,63. Coulston, F., Beyler, A. L., and Drobeck, H. F. (1960). Toxicol. A w l . Pharmacol. 2, 715.

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Davidaon, J. M., and Sawyer, C. H. (1961a). Proc. SOC.Ezptl. Biol. Med. 107, 4. Davidson, 0. W. (1962). I n “Conference on Immuno-Reproduction” (A. Tyler and K. Laurence, eds.), p. 27. Population Council, New York. Davis, B. K. (1963). Nature 197,308. Demol, R., and Ferin, J. (1964). Intern. J. Fertility 9, 197. Duncan, G. W., and Lyster, S. C. (1963). Fertility Sterility 14, 565. Duncan, G. W., Stucki, J. C., Lyster, S. C., and Ledmicer, D. (1962). Proc. SOC. Exptl. Biol. Med. 109, 163. Eckstein, P., and Mandl, A. M. (1962). Endocrinology 71,964. Edgren, R. A,, and Carter, D. L. (1962). J. Endocrinol. 24,525. Egeberg, O., and Owren, P. A. (1903). Brit. Med. J . 1,220. Epstein, J. A., Kupperman, H. S., and Cutler, A. (1958). Ann. N . Y . Acad. Sci. 71, 660. Ferin, J. (1962). Acta Endocrinol. 39,47. Fox, B. W., Jackson, H., Craig, A. W., and Glover, T. D. (1963). J. Reprod. Fertility 5, 13. France, E., and Pincus, G. (1964). Endocrinology (in press). Fridhandler, L., and Pincus, G. (1964). Ann. Rev. Pharmacol. 4, 177. Garcia, C. R., and Pincus, G. (1964). Intern. J . Fertility 9, 95. Gaunt, R., Chart, J. J., and Renzi, A. A. (1963). Ann. R e v . Pharmacol. 3, 109. Goldrieher, J. W. (1964). Med Clin. N.Am. 48, 529. Goldzieher, J. W., Haus, L. W., and Hamblen, E. C. (1947). A m . J. Obstet. Gynecol.

54, 820. Goldrieher, J. W., Moses, L. E., and Ellis, L. T. (1962). J. A m . Med. Assoc. 180, 359.

Goldzieher, J. W., Martinez-Manatou, J., Livingston, N. B., Moses, L. E., and Rice-Wray, E. (1963). Western J . Surg. Obstet. Gynecol. 71, 187. Greep, R. 0. (ed.) (1963). “Human Fertility and Population Problems.” Schenkman, Cambridge, Massachusetts. Guillemin, R. (1964). Recent Progr. Hormone Res. 20, 89. Gunn, S. A., and Gould, T. C. (1956). Endocrinology 58,443. Gunn, 5. A., Gould, T. C., Ginori, S. S., and M o m , J. G. (1955). Proc. SOC.Exptl. B i d . Med. 88, 556. Gunn, S. A., Gould, T. C., and Anderson, W. A. (1961). Arch. Pathol. 71,274. Gum, S . A., Gould, T. C., and Anderson, W. A. D. (1963). A m . J. Pathol. 42, 685. Harper, M. J. K. (1962). J. E n d o c r i d . 24, XX. Harris, G. W. (1961). In “Control of Ovulation” (C. A. Villee, ed.), p. 56. Pergamon, New York. Hartman, C. G. (1959). Fertility Sterility 10, 177. Hayashida, T. (1963). J. Endocrinol. 28,75. Hecht-Lucari, G . (1984). Intern. J . FertiZity 9, 205. Heimes, N. E. (1963). “Medical History of Contraception.” Gamut, New York. Heller, C. G., and Clermont, Y. (1904). Recent Progr. Hornwne Res. 20,545. Heller, C. G., Laidlaw, W. M., Harvey, H. T., and Nelson, W. 0. (1958). Ann. N. Y . Acad. Sci. 71, 649. Heller, C. G., Moore, D. J., Paulson, C. A., Nelson, W. O., and Laidlaw, W. M. (1959). Federation Proc. 18, 1057. Heller, C. G., Moore,D. J., and Paulson, C. A. (1961). TozicoZ. A w l . Pharnacol. 3, 1.

DRUGS USED IN CONTROL OF REPRODUCTION

31 1

Heller, C. G., Flageolle, B. Y., and Matson, L. J. (1963). Exptl. Mol. Pathol. Suppl. 2, 107. Holmes, R. L., and Mandl, A. M. (1962a). J. Endocrinol. 24,497. Holmes, R. L., and Mandl, A. M. (1962b).Lancet 11, 1174. Jackson, H. (1959).Pharmacol. Rev. 11,135. Jackson, H., Fox, B. W., and Craig, A.W. (1961).J. Reprod. Fertility 2, 447. Jackson, M.C. N. (1962).J. Endocrinol. 24,XXVI. Jackson, M. C. N. (1963).J. Reprod. Fertility 6, 153. Jackson, M. C. N., and Linn, R. (1964).Intern. J. Fertility 9, 75. Jochle, W. (1962).Angew. Chem. Intern. Ed. Engl. 1, 537. Johnson, V. E.,and Masters, W. H. (1963).Western J . Surg. Obstet. Gynecol. 71, 144.

Kanematsu, S., and Sawyer, C. H. (1963). Endocrinology 73,687. Kar, A. B., Dasgupta, P. R., and Das, R. P. (1961).J. Sci. Znd. Res. (India) 20C, 322. Kar, A. B., Bose, A. R., and Das, R. P. (1963).J. Reprod. Fertility 5,77. Katsh, S. (1962). In “Conference on Immuno-Reproduction.” (A. Tyler and K. Lawrence, eds.), p. 17.Population Council, New York. Klopper, A. (1961).Brit. Med. J . JI, 1354. Kobayashi, T., Takezawa, S., and Oshima, K. (1962). Endocrinol. Japon. 9, 302. Lakshman, A. B., and Nelson, W. 0.(1963).Nature 199,608. Langecker, H. (1961). Acta Edocrinol. 37, 14. Lauweryns, J., and Ferin, J. (1964).Intern. J. Fertility 9,s. Layne, D. S., Meyer, C . J., Vaishwanar, P. S., and Pincus, G. (1962).J. Clin. Endocrinol. Metab. 22, 107. Layne, D. S., Golab, T., Arai, K., and Pincus, G. (1963). Biochem. Pharmacol. 12, 905. Lipschuts, A., Inglesias, R., and Salinas, 8. (1983).J. Reprod. Fertility 6, 09. Loraine, J. A. (1984).Intern. J. Fertility 9,155. Loraine, J. A., Bell, E. T., Harknem, R. A., Mears, E. and Jackson, M. C. N. (1963). Lancet I& 902. Lostroh, A. J., Johnson, R., and Jordan, C. W., Jr. (1963). Acta Endocrinol. 44, 536. Lunenfeld, B. (1964).Intern. J. Fertility 9, 167. Lunenfeld, B., Sulimovici, S., and Rabau, E. (1963). J. Clin. Endocrinot. Metab. 23, 391. McCann, S. M., and Ramirez, V. D. (1964).Recent Progr. Hormone Res. 20, 131. MacLeod, J. (1961).Anat. Record 139,250. MacLeod, J., Sobrero, A. J., and Inglis, W. (1961).J. Am. Med. Assoc. 176, 427. Makepeace, A. W., Weinstein, G. L., and M. H. Freedman (1937).A m . J. Physiol. 119, 512. Mammen, E. F., Aoki, N., Oliveira, A. C., Barnhart, M. I., and Seegers, W. H. (1963).Intern. J . Fertility 8,653. Mann, T. (1954).“Biochemistry of Semen.” Wiley, New York. Mann, T. (1958).Studies on Fertility 9, 3. Matsumoto, S., Ito, T., and Inoue, S. (1SaO). Geburtsh. Frauenheilk. 20, 250. Mears, E. (1964a). Intern. J. Fertility 9,1 . Mears, E.(1964b). Proc. R w . SOC.Med. 53,204. Metcalf, M., and Beaven, D. W. (1963).Lancet II,1095. Michael, R. P. (1962). Intern. Congr. Physiol. Sci. 89nd, Leyden, 1968, Symp. XIV p. 650.Excerpta Medica Foundation, New York.

312

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Q.

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Moore, D. J., Roscoe, R. T., Matson, L. J., and Heller, C. G. (1962). Clin. Res. 10, 88.

Nelson, W. 0. (1941).Anat. Record 79,48. Nelson, W.O.,and Bunge, R. G. (1957).J. Urol. 77, %5. Nelson, W. O.,and Patanelli, D. J. (1960).Acta Endocrinol. Suppl. 51,905. Nelson, W. O.,and Steinberger, E. (1953).Federation Proc. 12, 103. Nikitovitch-Winer, M. B. (1962).Endocrinology 70, 350. Ostergaard, E.(1964).Intern. J. Fertility 9, 25. Paget, G. E., Walpole, A. L., and Richardson, D. N. (1961). Nature 192, 1191. Pariiek, J. (1960).J. Reprod. Fertility 1, 294. Parkes, A. S. (ed.) (1950).“Marshall’s Physiology of Reproduction,” Vol. 11. Longmans, Green, New York. Patanelli, D. J., and Nelson, W. 0. (1964). Recent Progr. Hormone Res. 20, 491. Paulsen, C. A.,Moore, D. F., Roscoe, R. T., and Heller, C. G. (1960). Acta Endocrinol. Suppl. 51, 203. Pelgram, L. 0 . (1964).Brit. Med. J. 1, 883. Pincus, G. (1955).Proc. 5th Inter. Conf. Planned Parenthood, Tokyo, 1956 p. 175. Excerpta Medica Foundation N. Y. Pincus, G. (1956).Acta Endocrinol. Suppl. 28, 18. Pincus, G. (1961). Jn “Modern Trends in Endocrinology” (Gardner-Hill, H., ed.), 2nd series. p. 231.Butterworths, London. Pincus, G. (1964).Advan. Chem. Ser. (in press). Pincus, G., and Chang, M. C. (1953).Acta Physiol. Latinoam. 3, 177. Pincus, G., Rock, J., and Garcia, C. R. (1958).Ann. N . Y . Acud. Sci. 71,677. Pincus, G., Garcia, C. R., Paniagua, M., and Shepard, J. (1962). Science 138, 439. Purshottam, N. N., Mason, M. M., and Pincus, G. (1961).Fertility Sterility 12, 346. Reichlin, S. (1963).New Engl. J. Med. 269, 1182. Rice-Wray, E., Aranda-Rosaell, A., Maqueo, M., and Coldrieher, J. W. (1963). Am. J. Obstet. Gynecol. 87, 429. Rock, J., Garcia, C. R., and Pincus, G. (1957).Recent Progr. Hormone Res. 13, 323. Roosen-Runge, E.C. (1962). Biol. Rev. Cambridge Phil. Soe. 37,343. Rumke, P. L. (1959). Immunopathol. Intern. Symp., l s t , BasellSeelkberg 1958 p. 145. Schwabe, Basel. Sawyer, C. H., and Kawakame, M. (1961). In “Control of Ovulation” (C. A. Villee, ed.), p. 79.Pergamon, New York. Searle, G. D. and Co. (1962).Proceedings of a Conference “Thromboembolic Phenomena in Women.” Chicago, Illinois. Sepal, S. J., and Nelson, W. 0. (1958).Proc. SOC.Exptl. Biol. Med. 98, 431. Senyal, S. N.(1962).Bull. Calcultta School Trop. Med. 10,s. Shelesnyak, M. C. (1956).Acta Endocrinol. 23, 151. Shelesnyak, M. C., Lunenfeld, B., and Honig, B. (1983). Life Sci. 1, 73. Sobrero, A. J., Fenichel, R. L., and Singher, H. 0. (1963).J . A m . Med. Assoc. 185, 136. Staemmler, H. J. (1960). In “Modern Developments in the Gestagen Field” (H. Nowakowski, ed.), p. 40.Springer, Berlin. Swyer, G. I. M. (1964). Proc. Roy. SOC.Med. 53, 210. Tietre, C.(196Oa).Proc. Rudolf Virchow Med. SOC.City N . Y . 19, 28. Tietse, C. (1960b). “Selected Bibliography of Contraception, 1940-1960.’’ Natl. Comm. Maternal Health, New York. Tietre, C. (1963).“Selected Bibliography of Contraception, Suppl. 1960-1963.’’Natl. Comm. Maternal Health, New York.

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Tyler, A. (1961). J. Reprod. Fertility 2, 473. Tyler, A., and Laurence, K. (eds.) (1982). “Conference on Immuno-Reproduction.” Population Council, New York. Tyler, E. T. (1963). J. Am. Med Assoc. 1 8 6 4 7 . Tyler, E. T. (1964). J. Am. Med. Assoc. 187, 562. Vanek, R. (1964). Intent. J . Fertility 9, 129. Villee, C. A. (ed.) (1961). “Control of Ovulation.” Pergamon, New York. Wallach, E. E., Garcia, C.-R., Kistner, R. W., and Pincus, G . (1963). Am. J . Obstet. Gynecol. 87, 991. Walser, H. C., Margolis, R. R., and Ladd, J. E. (1964). Intern. J. Fertility 9, 189. Walsh, E. L., Cuyler, W. K., and McCullagh, E. E. (1934). Am. J. Physiol. 107, 508. White, I. G. (1955). Australian J. Exptl. Bid. Med. Sci. 33,359. Young, W. C. (ed.) (1961). “Sex and Internal Secretions.” Williams & Wilkins, Baltimore, Maryland. Zaiiartu, J. (1964). Intern. J. Fertility 9, 225. Zeilmaker, G. M., and Carlsen, R. A. (1962). Acta Endocrinol. 41,321. Zuckerman, S. (ed.) (1962). “The Ovary,” Vols. 1 & 2. Academic Press, New York.

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Author Index Numbers in italics indicate the pages on which the complete reference8 are listed.

A Abadie, S. H., 159, 160, 166 Abercrombie, G.F.,246,262 Abramson, D.I., 40, 79 Abreu, B. E.,195, 969 Acheson, G.H.,242,969 Adam, B., 197,969 Adamson, D.W., 278,281 Adam-Ray, J., 170,269,963 Adey, W.R., 85, 86, 119,f26 Adler, T.K., 267, 268,983 Afonso, S.,2, 34,35, 38,80 Agate, F. J., 89, 122 Agostoni, E.,169, 963 Ahlquist, R. P., 174, 176, 177, 178,963 Ahmed, A., 99, 100, 103, 119 Akawie, R.J., 181,263 Albert, A., 252,963 Albertaon, N. F., 280,981 Aldinger, E. E.,34, 35, 37, 77 Aldunate, J., 239,969 Alella, A., 7,8, 9, 11, 76 Allan, W.,105, 119 Allen, D.A., 197,962 Allen, M.L., 231,960 Alles, G.A., 241, 9669 Alpert, M.,116, 192 Amin,A. H.,133,169, 217,963 Ananenko, E.,201, 269 Anderson, F.S., 4, 81 Anderson, W.A., 294, 310 Anderson, W.A. D., 293, 310 Andrews, M.C., 303, 309 Andrews, W.C., 303, 309 Angel, R. W.,87, 193 Aoki, N.,302,311 Apostolakia, M.,288, 6509 Aprison, M.H.,113, 191 Arai, K.,303, 311 Aranda-Rossell, d.,301,6519 Archer, S.,251, 966,280, 981

Arduini, A., 220, 960 Arilns, E.J., 176, 177, 178, 200,204, 963 Armitage, A. K.,99, 194 Armstrong, M.D., 182, 248,9665,961 Arnold, A.,251,966 Arnould, F.,110, 196 Artemov, N. M.,132,162 Arunlakshana, O.,246,963 Asads, S.,31, 55, 76 Asai, K.,87, 119 Astley Cooper, H.,95, 119 Athreya, B. H.,93,193 Augustinsson, K.B., 170,9669 Axelrod, J., 117, 119, 172, 179, 180, 182, 206, 216,222,223,246,248,249, 250, 251, 9669,960, 964,966,268,981 Axehon, J., 206,963 Ayala, G.F.,219, $68 Ayd, F. J., Jr., 105, 107, 109, 119 Aylesworth, R. J., 99, 118, 193 Azarnoff, D.L., 171,863

B Bacher, J. A., Jr., 135, 186 Bachmann, F., 179,980 Bacq, Z. M., 131, 132, 189, 173, 206, 214, 222, 963 Baird, H. W., 94, 196 Bakay, L.,227,963 Baker, W.W.,96, 98, 100,119 Balms, T.,64, 77,80 Baldwin, E.,134, 137, 188 Ballard, F.B., 14, 20,21,22, 25, 76, 77 Ballinger, W.F.,12, 24,75, 76 Balourdaa, T.A,, 15, 46,81 Balzer, H., 115, 184, 182,966 Ban&, U.K.,306,309 Barmde, R., 106, 191 Barbeau, A., 99, 113, 115, 116, 117, 119 Barcroft, H.,225, 363 Bardhmabmdya, S.,18, 77 315

316

AUTHOR INDEX

Barger, G., 168, 200, 201, 205, 243, 263 Barlow, C. F., 225, 264 Barnhart, M. I., 302, 311 Barraquer-Bordaa, L., 87, 119 Barron, A., 110, 1 f 9 Barron, K. D., 88, f26 Barsa, J., 107, 127 Barsky, C. A., 248, 260 Barsky, J., 249, 866 Bartholomew, A. A., 242, 263 Bass, A., 224, 263 Baas, H., 207, 269 Baum, H., 235,263 Bauat, W., 220, 263 Beach, H. D., 273, 281 Beaulnes, A., 222, 266 Beaven, D. W., 303, Slf Beckering, B., 110, 119 Beecher, H. K., 242, 263,274, 282 Beek, H., 106, 121 Beernink, K. D., 135, 136, 162 Beguin, M., 110, I23 Bein, H. J., 106, 119, 211, 212, 264 Bein, J., 245, 246, 263 Beiter, E. H., 161, 166 Bell, E. T., 299, 305, 309,Sf1 Belleau, B., 175, 176, 177, 178, 204, 207, 210, 229, 242, 243, 264 Bellet, S., 30, 31, 43, 46, 81 Bellville, R. E., 269, 282 Benda, C. E., 86,119 Benitea, H.,118, 119 Bennish, A., 69, 77 Benson, W. M., 281, 881 Bente, D. G.,106, 119 Bentley, K. W., 275, 2881 Berglund, E., 9, 12, 13, 19, 46, 77,81 Berk, M. S., 33, 65, 78 Berlin, D. D., 27,77 Berman, E. R., 249, 266 Bernard, C., 281, 881 Bernard, U., 30, 77 Berne, R. M., 7, 9, 10, 14, 16, 17, 18, 19, 23, 24, 31, 44,46, 77,78 Bernhang, A. M., 97, 119 Bernheimer, H., 116, 117, 119 Bertichamps, A., 107, 121 Bertler, A., 115, 119, 171, 181, 182, 211, 264, 266

Betham, E. J., 140, 141, 162 Bethlem, J., 113, 190, ldf, 124

Beuren, A., 13, 25, 46, 77,7